Cowie,  D. B.

Hershey,  A. D.

Cold Spring Harbor Laboratory Institutional Repository

VOL. 53, 1965 GENETICS: COWIE AND HERSHEY 572 Forsman, W. C., and R. E. Hughes, J. Chem. Phys., 38, 2118, 2123, and 2130 (1963).3 Higuchi, W. I., J. Phys. Chem., 65, 487 (1961).4 Frisch, H. L., J. Phys. Chem., 59, 633 (1955).6 Simha, R., H. L. Frisch, and R. F. Eirich, J. Phys. Chem., 57, 584 (1953).6 Lifson, S., J. Chem. Phys., 40, 3705 (1964).7 Zimm, B. H., J. Chem. Phys., 33, 1349 (1960).8 Lifson, S., and B. H. Zimm, Biopolymers, 1, 15 (1963).9 Goodrich, F. C., J. Chem. Phys., 22, 588 (1954).10 An expression similar to equation (21) was derived by an exact matrix method for finite nby S. Lifson and A. Roig [J. Chem. Phys., 34, 1963 (1961)].An expression similar to equation (28) was derived by Zimm7 in his treatment of DNA.12Jacobson, H., and W. H. Stockmayer, J. Chem. Phys., 18, 1607 (1950).18 Kuhn, W., Kolloid Z., 76, 258 (1936).14 Pouchly, J., CoU. Czech. Chem. Corn., 28, 1804 (1963).15Hoeve, C. A. J., E. A. DiMarzio, and P. Peyser, to be published.MULTIPLE SITES OF INTERACTION WITH HOST-CELL DNAIN THE DNA OF PHAGE X*BY DEAN B. COWIE AND A. D. HERSHEYDEPARTMENT OF TERRESTRIAL MAGNETISM, CARNEGIE INSTITUTION OF WASHINGTON,AND GENETICS RESEARCH UNIT, CARNEGIE INSTITUTION, COLD SPRING HARBOR, NEW YORKCommunicated November 4, 1964During lysogenization of Escherichia coli by phage X, the DNA of the phage isincorporated into that of its host to form a stable, compound chromosome. Themechanism of incorporation is unknown, but presumably calls for interactions atsites of complementary base sequence in the two DNA's. The existence of suchsites of interaction was demonstrated by Cowie and McCarthy' and by Green.2The isolation of specific parts of the X DNA molecule' 4makes possible attemptsto determine the intramolecular positions of the reactive sites. In principle, geneticdeletions can be utilized for the same purpose. Preliminary experiments along bothlines are reported in this paper.Materials and Methods.-Bacterial DNA was prepared from E. coli strain B(Berkeley) or W3110 (sensitive to phage X) by the method of Marmur.5 DNA fromphage X, genotype cb+ unless otherwise stated, labeled with p32 at 1-10 mc/mg P,was prepared according to Burgi.6 Interaction of the two DNA's was measured bythe DNA-agar method of Bolton and McCarthy.7 The solvent used, known asstandard saline-citrate (SSC), contains 0.15 M NaCl and 0.015 M sodium citrate.The terms 2 X SSC, SSC/30, and SSC/100 signify multiple or fractional concentra-tions of both salts.Molecular ends of X DNA were prepared as follows.4 P32-labeled DNA wasreduced to approximate quarter-length fragments by mechanical stirring. The twoterminal quarters were then rejoined through their cohesive sites by heating thesolution (in 0.6 M NaCl) briefly to 750C and cooling it slowly.8 The joined frag-ments were separated from the others by subjecting the mixture to preparativecentrifugation through a density gradient of sucrose and recovering the faster-sedimenting band. The isolated terminal fragments were disjoined by heating the58 GENETICS: COWIE AND HERSHEY PROC. N. A. S.solution to 750C (in 0.1 M NaCI) and cooling it quickly. The mixture was nextpassed through a methylated serum albumin column operated at 40C.3 The frac-tions eluting first under a concentration gradient of NaCl contained "left" ends.Fractions eluting last contained "right" ends but were contaminated with left ends.Pooled fractions of either kind were purified further by a repetition of the thermal-joining and centrifugation steps, this time recovering fragments that failed to join,representing mainly the excess over numerical equality of left or right ends, respec-tively. Left and right ends are so called with reference to the genetic map,3 andare recognized physically by the fact that left ends contain more guanine and cyto-sine, and are consequently denser, than right ends.Molecular weights9 of the isolated materials varied between 0.26 and 0.32 of thatof intact X DNA (31 X 106). Owing to fractionation with respect to size in themethylated albumin column, the recovered left ends were somewhat shorter thanthe right ends. Purity, as judged by density analysis, was about 90 per cent forright ends, and higher for left ends, as shown in Figure 1.,0zDens;tyFIG. 1.-Bands formed at equilibrium in a density gradient of CsCl. Left ends of XDNA molecules (density 1.715, upper diagram) and right ends (density 1.706, lower dia-gram), each representing about 28% of the original molecular length. Reference bandsat the right are Bacillus subtilis phage SP8 DNA of density 1.742. X DNA preparationswere heated to 750C for 1 min, cooled in ice, and spun in CsCl at 50C to prevent spon-taneous joining of left- or right-end contaminants to the major fractions. Photographedafter 30 hr at 44,770 rpm.Agar containing denatured E. coli DNA was prepared according to Bolton andMcCarthy.7 P32-labeled X DNA, either unfractionated or the isolated molecularends, was reduced to fragments of about 400,000 daltons by passage through aFrench pressure cell or by sonic treatment. The fragments were denatured byheating in 2 X SSC, and mixed with DNA agar. The mixture was heated in sealedVOL. 53, 1965 GENETICS: COWIE AND HERSHEY 59tubes at 60'C for 18-24 hr to permit specific interactions to occur, and transferredto water-jacketed columns for analysis.The b+ region of lambda DNA, representing base sequences present in wild-typeX but missing in the deletion mutant Xb2, was isolated by absorbing out cross-reactingmaterial from P12-labeled Xcb+ DNA fragments on Xcb2 DNA agar. The fractionfailing to attach in two or more treatments contained 20-26 per cent of the totallabeled DNA. It was concentrated by evaporation, dialyzed against 2 X SSC, andcentrifuged to remove precipitated agar.Results.-Left and right molecular ends: The ability of X DNA and its molecularends to form specific complexes with E. coli DNA is illustrated in Table 1. Com-paring results presented in the individual rows, one sees that left ends, right ends,and unfractionated X DNA react about equally. Comparing vertical columns, onesees that the fraction of X DNA bound depends on the absolute and relative con-centrations of the two DNA's (as well as other variables) in the mixture. Thestrong dependence on the concentration of E. coli DNA in the agar is understand-able, since only 0.2 per cent of that DNA participates in the reaction. The fractionof X DNA participating is about 34 per cent.'TABLE 1SPECIFIC ATTACHMENT OF X DNA FRAGMENTS DERIVED FROM DIFFERENT PARTS OF THE MOLECULETO Escherichia coli DNA AGARI Labeled DNA Bound (%)E. coli DNA in agar Labeled X DNA X(fig) (jsg) Unfractionated X Left ends X Left ends20 (W3110) 0.02 12.7 8.3 8.0220 (W3110) 0.04 23.3 18.9180 (BB) 0.005 27.6 24.7 19.312(BB) 0.005 17.9 13.0 16.424 (BB) 0.005 18.1 12.1 17.4The mixtures contained 0.2 gm of DNA-agar and an equal volume of labeled DNA in 2 X SSC, andwere held for about 18 hr at 60'C, transferred to a column, and washed with ten 10-ml portions of 2 XSSC at 600C. The specifically bound, labeled DNA remaining in the agar was removed for assay by wash-ing with SSC/100 at 751C.The ability of the central portion of the X DNA molecule to react with E. coliDNA can be estimated by difference from the data of Table 1. Such estimatessuggest that the reactive sites may be appreciably more concentrated near thecenter of the molecule than near its ends. However, the measurements are biasedby the fact that, at equal DNA concentrations, the concentration of reactive frag-ments specified with respect to kind is higher in the fractionated than in the un-fractionated preparations. The only safe conclusion is that both terminal andcentral parts of the X DNA molecule contain reactive sites.The left end of the X DNA molecule contains about 56 per cent guanine andcytosine, and the right end about 47 per cent (Fig. 1 and unpublished data). Themelting temperatures of the two ends reflect the difference in composition. If thesequences interacting with E. coli DNA differ in composition in the same way asthe molecular parts in which they lie, one might expect fragmented left ends to meltout of E. coli DNA agar at a higher temperature than right ends.Thermal chromatograms presented in Figure 2 show in fact that the bonds bywhich the left ends of X DNA attach to E. coli DNA are more stable to heat thanthose involved at the right end. Both ends, however, show high- and low-meltingcomponents. The chromatogram for left ends exhibits maxima at 60, 66, and 720C,60 GENETICS: COWIE AND HERSHEY PROC. N. A. S.aK-Athat for right ends at 63 and 690C (in SSC/30).The chromatogram for unfractionated X DNA isUnfractiorted roughly a composite of the two extremes. ItDNA/ seems likely, therefore, that even within individualsections of the molecule there are multiple seg-5 * ments capable of interaction with E. coli DNA.,H10, The b2+ region: Another method of isolatingspecific parts of the DNA molecule depends on thec o ' ,my\T z/ Iuse of genetic deletions. The b2 deletion mutantE t I fJ '" (' - 5 t proved suitable for this purpose. The mutantphage contains a DNA molecule about 18 perC] ~ Left end A DNA cent shorter than that found in wild-type X.6, 10SO 8 The site of the deletion has not been determinedoUoB d directly, but the loss is believed to have occurred10 Unfractionated near the host-range (h) locus, to the left of themolecular center, in a region important forlysogeny.1 It is probably a region rich in adenineand thymine, since the mutant DNA contains adiminished proportion of these bases.12/Eja,a \ Isolation of the b2+-specific region of the XDNA molecule is described in the Materials and5 Methods section of this paper. Its interaction\/ _ with E. coli DNA is shown in Table 2. SinceRight end ADNA the material was not highly purified, competing!070 8°0 unlabeled b2 DNA was used to achieve theTemperature C desired specificity. The results show that theFIG. 2.-Thermal chromatograms b2+ region of the X DNA molecule, like the otherof complexes formed between E. coli regions tested, contains sites capable of reactingl da DNAioileculeparTshe mxd with E. coli DNA. Green2 and Kiger and Green"4DNA's were annealed at 600C in made the complementary test and found that Xb22 X SSC in the usual way, trans- DNA contains most of the sites reactive with E.ferred to a column, and washed withten 10-ml portions of 2 X SSC coli DNA present in X wild-type DNA. Theirfollowed by two portions of SSC/30 results left open the possibility that the b2+ regionat 500, each portion passing throughthe column in about 10 min. contains no sequences incommonwithE. coli.a single washewtDNhA re/3oved in The specificty of interaction: The specificity ofof a series of temperatures was interactions of the type studied here has beenmeasured. The results presented in checked repeatedly by Bolton and McCarthy andthe figure were obtained with agarcontaining about 100 ug DNA/gm. others. We found, in addition, that labeled XWhen agar containing DNA at DNA fragments do not bind to T5 DNA agar.higher concentrations was used,DNmuch of the labeled DNA eluted The finding of Cowie and McCarthy' that theat lower temperatures and charac interacting segments in the DNA's of X and E.coli are of about the same length signifies thatthere are unique complementary sequences present in the two DNA's. Thisconclusion was checked by applying labeled E. coli DNA fragments to X DNA agar,which showed no measurable attachment (theoretical expectation about 0.2%) inexcess of the small background seen with agar containing no DNA. Finally, thehigh melting temperature of the complexes formed by labeled X fragments in E. coliVOL. 53, 1965 GENETICS: COWIE AND HERSHEY 61TABLE 2SPECIFIC ATTACHMENT TO DNA AGAR OF NUCLEOTIDE SEQUENCES (Xb+ SEGMENT) PRESENT IN XWILD-TYPE BUT NOT IN Xb2 DNADNA in AgarLabeled DNA Xb+ Xb2 E. coli W3110Unfractionated X 79 73 13Xb+ segment 72 26 9Xb+ segment* 55 11 6Each test mixture contained 0.2 gm agar (10-20 jig DNA) + 0.002 jg P32-labeled DNA, shearedand denatured.The numbers express % of labeled DNA not eluted by 2 X SSC at 600C.* Each test mixture contained in addition 1 ug (5 ;&g in other experiments) of unlabeled, sheared,and denatured Xb2 DNA as competitor.DNA agar (Fig. 2), similar to that found for X-X DNA complexes, signifiesauthentic matching of numerous base pairs. Kiger and Green14 reached the sameconclusion after studying the thermal stability of the appropriate DNA-RNA com-plexes.Discussion and Conclusion.-The homology test of Bolton and McCarthy as em-ployed in this work consists in reducing lambda DNA molecules to about 150single-stranded fragments and determining what fraction of them can attach todenatured E. coli DNA.The previous experiments' had shown that about one third of them can attachunder optimal conditions. The present results show that the 50 or so reactive frag-ments are derived more or less equally from the left, right, and central quarters ofthe molecular length. In addition, the 18 per cent stretch in X wild-type DNA cor-responding to the b2 deletion yields reactive fragments. Finally, analysis in termsof melting temperature of the complexes further subdivides the fragments derivedfrom any one section of the molecule into two or three subclasses. The inferenceis clear that there are at least three and probably more sites on the X DNA mole-cule that can interact with E. coli DNA. However, the actual number, length,and distribution of these sites remains to be determined.The results described do not throw much light on questions concerning the originand function of the homology between X DNA and the DNA of its host, nor aboutthe role of the common sequences in lysogeny. One possibility can be excluded,however. The stretches involved cannot belong to a class of periodically repeatedsequences present in both DNA's, because in the DNA of E. coli they are relativelyvery rare. It should prove instructive, therefore, to ascertain the location of thecross-reacting sites in the genome of E. coli.* This investigation was supported by USPHS research grant HD-01228 from the NationalInstitute of Child Health and Human Development.1 Cowie, D. B., and B. J. McCarthy, these PROCEEDINGS, 50, 537 (1963).2 Green, M. H., these PROCEEDINGS, 50, 1177 (1963).3Hogness, D. S., and J. R. Simmons, J. Mol. Biol., 9, 411 (1964).4Hershey, A. D., and E. Burgi, unpublished.6 Marmur, J., J. Mol. Biol., 3, 208 (1961).6 Burgi, E., these PROCEEDINGS, 49, 151 (1963).7Bolton, E. T., and B. J. McCarthy, these PROCEEDINGS, 48, 1390 (1962).8 Hershey, A. D., E. Burgi, and L. Ingraham, these PROCEEDINGS, 49, 748 (1963).9 Burgi, E., and A. D. Hershey, Biophys. J., 3, 309 (1963).ie Kellenberger, G., M. L. Zichichi, and J. Weigle, J. Mol. Biol., 3, 399 (1961).1' Zichichi, M. L., and G. Kellenberger, Virology, 19, 450 (1963).62 GENETICS: GARTNER AND ORIAS PROC. N. A. S.12Hershey, A. D., E. Burgi, F. R. Frankel, E. Goldberg, L. Ingraham, and G. Mosig, CarnegieInst. Wash. Yr. Bk., 62, 481 (1963).13Miyazawa, Y., and C. 4. Thomas, Jr., personal communication.14Kiger, J., and M. H. Green, Virology, 22, 602 (1964).PLEIOTROPIC EFFECTS OF SUPPRESSORS OF A LAC-"OPERATORNEGATIVE" MUTATION IN ESCHERICHIA COLI*BY T. K. GARTNER AND E. ORIASDEPARTMENT OF BIOLOGICAL SCIENCES, UNIVERSITY OF CALIFORNIA (SANTA BARBARA)Communicated by Katherine Esau, November 12, 1964A suppressor mutation is a genetic change which removes the mutant phenotypeassociated with another mutation, and which is separable from the first mutationby recombination. Of particular interest are suppressor mutations which act atthe level of translation of the gene modified by the first mutation. In this con-text, it is useful to consider if the lesion occasioned by the original mutation leadsto the formation of a mis-sense or nonsense triplet. A mis-sense triplet results in anamino acid substitution,' while a nonsense triplet results in an untranslatable codon.2Extragenic suppression of a mis-sense triplet has been demonstrated for an allele ofthe tryptophan synthetase system' of Escherichia coli. In the same organism,extragenic suppression of a nonsense triplet has been demonstrated for certainambivalent alleles of the A cistron of the rII system of phage T4,1 for certain ambermutants of the cistron which codes for the head protein of T4,4 for a mutant (020)allele of the fl-galactosidase structural gene,5' 6 and for a mutant allele of the alka-line phosphatase structural gene.7The mechanism(s) of suppression of mis-sense or nonsense triplets has not beendemonstrated experimentally. It has been speculated that alterations of either anactivating enzyme or a transfer-RNA could account for suppression.8 More re-cently, ribosomes have been implicated as another component whose alteration canresult in suppression.9 Needless to say, alteration of any component of the trans-lation system which does or can interact with messenger-RNA during the trans-lation process might result in suppression. Whatever the mechanism, it might beexpected that alteration of the translation machinery of a cell would, in certain in-stances, affect the translation of genetic information other than the original mu-tant triplet.3 This type of alteration of the translation machinery could be indi-cated by an unusual physiological behavior of a strain harboring the responsible sup-pressor mutation.In this paper, we report the properties of certain mutants capable of suppressingthe lac- phenotype determined by the "operator negative" mutations in strain2320(X) of E. coli. The suppressors were selected for further study because oftheir concomitant acquisition of (a) the ability to suppress a set of rII mutants ofphage T4 and certain mutants of T7,6 11 and (b) a variety of new physiological prop-erties not obviously related to suppression of the lac- phenotype. Suppression ofthe lac- phenotype is thought to be accomplished by the acquisition of the ability totranslate the nonsense triplet in the 020 allele in strain 2320(X).6 12 Pleiotropic ef-

Multiple Sites of Interaction with Host-Cell DNA in the DNA of Phage Lambda

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