JOURNAL OF BACTERIOLOGY, Nov. 1973, p. 656-662 Vol. 116, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Deletion Mapping and Orientation of the Operon of Escherichia coli on a Transducing Bacteriophage ALESSANDRA AVITABILE, CARMELO B. BRUNI, MARIA S. CARLOMAGNO-CERILLO, MARYLIN MEYERS, GERMANA VIGLIAR, AND FRANCESCO BLASI Centro di Endocrinologia ed Oncologia Sperimentale del CNR, IIIstituto di Patologia Generale, 2a Facolt& di Medicina, Via Sergio Pansini 5, 80131, Naples, Italy, and Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland 20014 Received for publication 31 July 1973 The defective prophage 080icI857dhis has been mapped through both marker rescue and deletion analysis. Deletions have been isolated which put residual his genes close to trp genes. Analysis of these deletions shows that the histidine operon on the prophage is oriented clockwise as on the bacterial chromosome, thus opposite to the orientation of the trp operon. The presence of the his promoter-operator region is inferred by the ability of the prophage-carrying strain to derepress sequentially under conditions in which the histidine concen- tration is limiting. In addition to his, the gnd gene is also present on the prophage and is located between his and trp operons. The bacterial genes are inserted in the right arm of the prophage and substitute for all of the late function genes, except for the first three. These data indicate that the "sense" strand for transcription of the his operon in vivo must be the "R" strand.

A specialized transducing phage carrying the strain, and 480h is a host-range mutant which can entire Escherichia coli K-12 histidine operon, grow on tonB hosts (10). The symbol 480iX indicates a O8OiXc1857dhis (480dhis) was recently isolated hybrid phage (480 and lambda) which carries the is immunity region of lambda (19) with a temperature- in our laboratory (2). The transducing phage sensitive repressor mutation (c ,857). Phage 480 defective (2), which is not surprising since the amber lysates were obtained from R. E. Wolf, Jr., and his operon is made up of about 11,000 nucleo- had been prepared and titrated on a supD strain (CA tide pairs (4). In view of the use of 080dhis in 5013). Transduction was carried out as described by studies of the regulation of the expression of the Adams (1). his operon conducted in vitro, we have carried Solid and liquid media. The media employed have out a more detailed genetic and physiological been described previously (2). study of 480dhis lysogens with the aim of Enzymatic assays. The cells from a 200-ml culture answering the following questions: (i) is the his were harvested at an optical density of 0.650 at 650 nm, centrifuged at 4 C, washed with 0.05 M tris(hy- operon of 080dhis under histidine control? (ii) droxymethyl)aminomethane (Tris) buffer, pH 7.8, does 480dhis carry bacterial genes other than suspended in 5 ml of the same buffer, and passed his? (iii) which region of the phage genome has through a French pressure cell at 12,000 psi. The been replaced by the bacterial genes? and (iv) extract was centrifuged for 45 min at 30,000 x g at what is the direction of transcription of the his 4 C, passed through a column (1 by 7 cm) of Sephadex operon on the prophage genome, and thus which G-50 coarse (washed and eluted with 0.05 M Tris one of the phage strands acts as a template in buffer, pH 7.8), and assayed for and for the in vivo transcription of the his operon? enzymatic activities. Protein concentration was esti- mated by the method of Lowry et al. (13), with bovine serum albumine as standard. The activity of gluco- MATERIALS AND METHODS nate-6-phosphate (EC 1.1.1.44) was Bacterial strains. The strains used in this study assayed by measuring the increase in reduced nicotin- are described in Table 1. Genotype and phenotype amide adenine dinucleotide phosphate (NADPH) symbols follow the nomenclature of Demerec et al. absorption at 340 nm dependent on 6-phosphogluco- (6). nate and cell extract (7). Assays of the histidine Phage strains. The phage strains used were 080 and definition of enzymatic units have been and several of its mutants. Phage 480v is a virulent described by Martin et al. (15). 656 VOL. 116, 1973 ORIENTATION OF his OPERON ON 080dhis 657 TABLE 1. Bacterial strains Strain J Genotypea | Source and/or reference Escherichia coli K-12 SB1801 his gnd deletion, rha2A, mal, lambdar P. Hartman (2) FB53 SB1801 lysogen for 080iA and 4680iAdhis (2) FB73 SB1801 lysogen for 480i' dhis (2) FB78 his FIE71 gnd tonB trp Derived from FB73; FB82 his (H)AFIE72 gnd tonB trp spontaneously re- FB88 his GDCBHAFIE73 gnd tonB sistant to 480v FB89 his GDCBHAFIE74 gnd tonB trp and colicin V, B. DF710 F- edd gnd tyrA pyrD thi str D. G. Fraenkel (16) RW84 F- pgi edd eda (gnd his deletion) str R. E. Wolf, Jr. CA5013 supD R. E. Wolf, Jr. Salmonella typhimurium LT2 TR48 F' hisDa2378/hisBE612 trpA8 purE801 J. C. Loper (8) SC419 F' hisG5961/hisOF644 trpl30str J. C. Loper (8) SC7 F' hisB1205/hisOF644 trpl3O str J. C. Loper (8) SC83 F' hisA5925/his0F644 trpl3O str J. C. Loper (8) SC41 F' hisF5971/hisOF644 trpl30 str J. C. Loper (8) a Genetic symbols for E. coli according to Taylor and Trotter (21) and for S. typhimurium according to Sanderson (17).

Isolation of deletions of 80dhis prophage. A RESULTS 5-ml culture of FB73 in Z broth was centrifuged, and the cells were resuspended in 0.1 ml of 080v lysate Presence of gnd on 480dhis. The episome (1012 plaque-forming units [PFU]/ml) and 0.4 ml of (F'... 402 his+) which has been integrated at colicin lysate (10). This bacterial suspension was tonB as a step preliminary to the isolation of incubated at 34 C for 15 min, and samples were 480dhis (2) carries the structural gene for glu- spread on Z plates. After 24 to 48 h at 34 C, survivors conate-6-phosphate dehydrogenase (gnd) (R. E. were purified by streaking on Z plates. The survivors Wolf, Jr., personal communication). were tested by replica-plating for resistance to 480v To assess whether the gnd gene is also present and colicin, for sensitivity to 80h, and for auxotro- phy. Auxotrophy was tested for requirement of histi- on 080dhis, strain RW 84 was transduced to dine and tryptophan, and for the ability to grow on His+ with a lysate from strain FB53 (double histidinol. Histidinol is the immediate precursor of lysogen for 080 and 480dhis). His+ transduc- histidine and its utilization requires a functionally tants were checked for ability to grow on gluco- active histidinol dehydrogenase (EC 1.1.1.23; nate as sole carbon source. The recipient RW84 of the second structural gene, the hisD gene [4]). (his, gnd, edd) cannot grow on gluconate as sole Marker rescue with 080 amber mutants. FB 73 carbon source, since it lacks both gluconate- was grown at 34 C in minimal citrate medium (22) 6-phosphate dehydrogenase (gnd) and Entner- supplemented with 0.2% glucose and induced at 41 C Doudoroff gluconate-6-phosphate dehydrase for 15 min. A loopful of induced FB73 was streaked on (EC 1.1.1.43; edd). Either one of these two a lawn of SB1801, and lysates of the different 080 enzymes is necessary for the utilization of amber mutants (106 to 8 x 108 PFU/ml on strain CA5013) were cross-streaked. The presence of an area gluconate (16; R. E. Wolf, Jr., personal commu- of lysis at the confluence of the streaks was taken as nication). Transductants were selected on mini- an indication of recombination between 480dhis and mal plates supplemented with glucose, purified, 480 amber mutants. and then tested for growth on minimal plates Pattern of derepression of the his operon. Strain supplemented with 0.5% gluconate. All of eight FB73 grown at 34 C in minimal citrate (22) with 0.2% transductants tested (from two individual ly- glucose was derepressed by the addition of thiazolo- sates) were able to grow on gluconate as sole D,L-alanine (final concentration, 1 mM). Samples carbon source. were withdrawn at various times before and after The presence of gnd in the genome of 080dhis derepression, and the specific activity of several of the was confirmed by enzymatic assays. Gluconate- histidine enzymes was determined (15). The scheme of the experiment closely followed that described by 6-phosphate dehydrogenase activity is absent in Berberich, Venetianer, and Goldberger (3). a strain (SB1801) containing a total deletion of The levels of strain FB82 were measured on his and gnd genes, but can be detected in the cells cultured in minimal citrate (22) with 0.2% same strain after lysogenization with k80dhis. glucose, supplemented with tryptophan (30 ug/ml) In fact, strain FB73 (SB1801, singly lysogenic and histidine (20 jg/ml) or histidinol (150 ig/ml). for 480dhis) contains roughly wild-type levels of 658 AVITABILE ET AL. J. BACTERIOL. gluconate-6-phosphate dehydrogenase, whereas ture-sensitive (showing that they still contain the parental strain, SB1801, has no detectable the temperature-sensitive lambda repressor enzyme activity. The results of these experi- (c,857)), tonB (because of the selection used), ments are presented in Table 2. This activity is and His- (a property which has been scored missing in all three classes of his mutants (see for). Furthermore, classes I and III are Hol- below). (unable to use histidinol instead of histidine), Derepression of the histidine operon car- whereas class II mutants are Hol+. Classes II ried by 48Odhis. As shown in Fig. 1, addition of and III, finally, are also tryptophan auxotrophs. thiazolo-D,L-alanine, an inhibitor of the first This last requirement cannot be satisfied by enzyme (14), to strain FB73 (single lysogen for indole, showing that the deletion does not end k80dhis) slows down the growth rate and results in the trpA gene, but continues into trpB or in derepression of the histidine enzymes. In Fig. further (24). 1, the derepression pattern for the enzymes The activity of several histidine biosynthetic encoded in the G, B, and A genes is shown. As enzymes was assayed in crude extracts of the expected from the data on Salmonella (3), the mutant cells representative of each class (Table pattern of derepression is sequential and follows 2). Mutants of classes I and III do not have any the relative position of the genes in the histidine of the enzymatic activities tested. In contrast, operon as determined genetically (8, 9). Meas- class II mutants appear to contain enzymatic urements of the C and F gene products, al- activities encoded in the operator-proximal though not shown in Fig. 1, fit very well into the genes and to be lacking those activities encoded sequential pattern. in the operator-distal part of the his operon. On It is known that Salmonella his strains, still this basis, for instance, mutant FB82 appears to having a functional hisD gene, derepress when carry the first four structural genes of the his grown on histidinol (3). Under these conditions, operon (hisG, D, C, and B), but not the last four derepression also occurs in the deletion mutants (hisA, F, I, and E). The presence of the hisH of class II (next section). The results are re- gene, which is located between these two groups ported in Table 3. of genes, has not been determined. Properties of 080dhis prophage deletions. Mutants representative of each class have To determine the orientation of his on the been crossed with Salmonella strains carrying prophage map, we have isolated a set of dele- E. coli F'his episomes. The cross was carried out tions starting outside of the prophage and on glucose minimal plates in the absence of extending into the histidine operon to various histidine. His+ merodiploids were found only distances. These deletions have been isolated in when mutants of class II were used as recipients a strain (FB73) singly lysogenic for .80dhis (2), in the cross. In this case, complementation among survivors of the treatment with 480v and could be found only with F'his mutations proxi- colicins V and B (10, 11). Thirty independent mal to the operator end of the his operon. The tonB mutants, resistant to 480v and colicins results of the crosses are summarized in Table 5. (10), still harbor the immunity region of the Marker rescue experiments. Marker rescue phage, but have lost the ability to grow on experiments were carried out crossing 480dhis, minimal medium. These mutants, grouped into present in the singly lysogenic strain FB73 three classes, have the properties summarized (lysogenic for 080dhis) with X80 amber mutants in Table 4. All classes of mutants are tempera- (Table 6). Only mutants in five of the late

TABLE 2. Specific activities of histidine biosynthetic enzymesa and gluconate-6-phosphate dehydrogenaseb in deletion mutants isolated from a strain singly lysogenic for 08Odhis Sp enzymatic act Strain hisG hisD hisC hisB hisA hisF hisl gnd SB1801 .0 0 0 0 0 0 0 0 FB73 (SB1801, 480dhis) 8.5 1.5 4.4 3.2 6.2 10.4 5.4 260.0 FB88 (class I) .NT NT 0 0 0 0 0 0 FB82 (class I). 7.9 2.2 2.8 5.7 0 0 0 0 FB89 (class III) .NT NT 0 0 0 0 NT 0 a Enzymatic assays and definition of enzymatic units are reported in reference 15. I Specific activity of 6-phosphogluconate dehydrogenase is expressed in nanomoles of NADPH+ produced per minute per milligram of protein. CNot tested. VOL. 116, 1973 ORIENTATION OF his OPERON ON 080dhis 659

.8 7 .6 .5 E .4 C

CoCD .3 LOw .2

.1

U) 4 c

L. I- 4..-0 -a 3

-._ -0- 2 ._u

._ In

-60 0 60 120 TIME (minutes) FIG. 1. Kinetics of derepression in FB73. The top of the figure shows the growth curve. The bottom shows the specific activity of three of the enzymes examined: 0, hisG (pyrophosphorylase); e, hisB (phosphatase); and 0, hisA (). The vertical line indicates time of addition of thiazolo-D, L-alanine (TA.). For the sake of clarity, overlapping of the lines has been avoided by adding a constant factor to each set of points. function genes of 080 appear to recombine with tive. In fact, the length of deoxyribonucleic acid 080dhis (genes 1, 2, 3, 17, and 18). These (DNA), corresponding to the his operon, has correspond to the A, W, B, Q, and R genes of been estimated to be about 11,000 nucleotide lambda (18; R. E. Wolf, Jr., personal communi- pairs (4). If one considers also the length of cation). DNA corresponding to the gnd gene and the region between the his and gnd genes, one could DISCUSSION roughly estimate about 15,000 nucleotide pairs In this paper, we have reported experiments for the length of bacterial DNA carried by the which have allowed the construction of a map of phage. Phage 080, on the other hand, is made 080dhis, previously isolated in our laboratory up of about 48,000 nucleotide pairs (23). Since (2). This phage carries the whole his operon and only a very limited extra amount of DNA can be gnd gene. This explains why 080dhis is defec- packaged in the lambda-like phages (12), 660 AVITABILE ET AL. J. BACTERIOL. TABLE 3. Level of histidine biosynthetic enzymes in out. FB73, in fact, harbors the temperature-sen- mutant FB82 grown in the presence of histidine or sitive lambda phage repressor mutation, c,857. histidinol These results indicate that the his operon car- Enzymatic activity' ried by 080dhis can be derepressed under condi- Mediuma tions of limiting histidine concentration and "C" "B" repressed under conditions of excess histidine, thus strongly suggesting that the operator-pro- Plus histidine ...... 2.4 6.4 moter region of the operon is carried on the Plus histidinol ...... 21.5 19.3 chromosome of k8Odhis. a Cells were cultured in glucose-citrate-minimal Taking into account the growth require- medium supplemented with tryptophan (30 Ag/ml) ments, the derepression data, the conjugation and histidine (20 ug/ml) or histidinol (150 ug/ml). b Enzyme assays were performed as described by TABLE 5. Complementation analysis of deletion Martin et al., and activities are expressed in enzyme mutants isolated from FB73a units per milligram of protein (15). Recipient Donors F' his TABLE 4. Classes of deletions isolated among survivors of the treatment of FB73 with 080v and Class Mutant SC419 TR48 SC7 SC83 SC41 colicins V, B ClassMutant (G- (D-) (B-) (A-) (F-) Phenotype I FB88 NT NT Classe II FB78 NT + + + _ Hise Holc Gndd TonB' Tip| Indole" 11 FB82 NT + + - - III FB89 - - NT NT NT 1(12) - - - - + NT' 11 (21) - + - - - - a The results are expressed in terms of the ability of fI (4) - - - - - a deletion mutant to complement mutations carried by the F' his- episome; i.e., the plus sign indicates aNumbers in parentheses refer to the number of ability of the merodiploid to grow on plates not independently isolated mutants in each class. supplemented with histidine. b Minus signs indicate a requirement of histidine.or " Not tested. tryptophan for growth. cAbility of histidinol (Hol) or indole to satisfy TABLE 6. Recombination of JaOamber mutants with growth requirement for histidine or tryptophan. __8Odhis prophagea d Determined by measurement of enzymatic activ- ity. 80amber Plaque-forming e TonB mutants (-) are resistant to 4s80v and 080 gene mutantmutant recombinants with sensitive to 080h. ~~80dhis ' Not tested. 1 KS3 + 2 KS1 + 480dhis must have lost a rather large portion of 3 KS6 + its genome. The results obtained in marker 3 KS51 + rescue experiments, in fact, show that 480dhis 4 KS36 _ is lacking most genes for the head and tail, with 5 KS10 _ the exception of the first three head genes 6 KS21 _ (Table 6, Fig. 2). 7 KS11 _ The his operon carried by the phage appears 8 KS2 _ in 9 KS4 _ to be under histidine control while the 10 KS14 _ prophage state. Both a defective lysogen for 11 KS7 _ 480dhis and deletions cutting for various 13 KS28 _ lengths into the his operon of the phage are able 14 KS8 _ to derepress sequentially upon addition of thia- 15 KS326 _ zolo-D, L-alanine. Comparison of the derepres- 16 KS258 _ sion data (Fig. 1) with those of Salmonella (3) 17 KS121 + shows a difference in the time when derepres- 18 KS91 + sion starts; the time of derepression and the a"Heat-induced FB73 was streaked vertically on a intervals between derepression of the different tryptone agar plate seeded with 108 cells of SB1801. A genes are shorter in Salmonella. These differ- loopful of 080 amber mutant lysates was cross- ences very likely depend on the rather slow streaked, and the plates were incubated at 37 C for 24 growth rate of strain FB73 and on the tempera- to 36 h. An area of lysis at the confluence of the ture (34 C) at which the experiment was carried streaks was taken as a positive result. VOL. 116, 1973 ORIENTATION OF his OPERON ON 080dhis 661

tdk 8OiX h ist idi ne oner-on gndTonB Trp cysB

FIG. 2. Prophage map of 08Odhis. The extent of the deletions isolated is reported at the bottom of the map. Continuous horizontal lines represent the portion of genetic material whose loss has been established experimentally. Interrupted lines indicate the extent of genetic material which could have been lost in the mutants, but whose absence has not been proven. Lambda genetic symbols are used for ,80 genes (see text). Location of other bacterial or phage markers is summarized in references 21 and 5, respectively. studies with F' his episomes, and the measure- ACKNOWLEDGMENTS ment of the enzymatic activities, it is possible to Part of this work was supported by grant 71.02388.54 of define the orientation of the his operon on the Consiglio Nazionale delle Richerche to C. B. Bruni under the Italy-USA Cooperation program. prophage map. Class I and III deletions appear We are very grateful to R. E. Wolf, Jr., for a gift of 080 to be lacking all the genes of the his operon as amber lysates, for bacterial strains, for useful suggestions, well as the gnd gene. Also class II deletions lack and for communicating unpublished results; to J. C. Loper for the gnd gene, but still retain some of the his a collection of his episomes; and to R. Favre, R. F. Goldberg- er, T. Klopotowski, and M. Iaccarino for helpful discussion genes. Thus, it appears that the order of genes is and useful criticism in writing this manuscript. The help of R. his, gnd, tonB, trp. Since in class II deletions Cerillo is gratefully acknowledged. the his genes which are missing are those distal to the operator (i.e., mutant FB78 still retains LITERATURE CITED GDCBHA genes, and mutant FB82 the [PO] 1. Adams, M. H. 1959. Bacteriophages, p. 454. Interscience retains the POGDCB genes), the order of genes Publishers, Inc., New York. can be set in more detail: c1857 his POGDC- 2. Avitabile, A., M. S. Carlomagno-Cerillo, R. Favre, and F. BHAFIE gnd tonB trp. The position of the c1857 Blasi. 1972. Isolation of transducing phages for the histidine and isoleucine-valine operons in Escherichia mutation must be to the left of his since all coli K-12. J. Bacteriol. 112:40-47. three classes of deletions still retain tempera- 3. Berberich, M. A., P. Venetianer, and R. F. Goldberger. ture sensitivity (due to the presence of the c,857 1966. Alternative modes of derepression of the histidine mutation). The marker rescue data (Table 6) operon observed in Salmonella typhimurium. J. Biol. Chem. 241:4426-4433. genes furthermore indicate that the bacterial 4. Brenner, M., and B. N. Ames. 1971. The histidine operon are inserted into the right arm of the prophage. and its regulation, p. 349. In H. S. Vogel (ed.), Figure 2 shows a schematic map of b8Odhis Metabolic pathways, vol. 5. Academic Press Inc., New prophage. York. in 5. Davidson, N., and W. Szybalsky. 1971. Physical and The relative orientation of his and trp the chemical characteristics of lambda DNA, p. 45-82. In lysogen of k80dhis (Fig. 2) is the same as on the A. D. Hershey (ed.), The bacteriophage lambda. Cold E. coli chromosome (20). A similar orientation Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and structure have been found in a phage 6. Demerec, A., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 1966. A proposal for a uniform nomenclature carrying gnd and his (R. E. Wolf, Jr., and D. G. in bacterial genetics. Genetics 54:61-76. Fraenkel, Fed. Proc., p. 444, 1972; R. E. Wolf, 7. Fraenkel, D. G., and B. H. Horecker. 1964. Pathways of Jr., personal communication). D-glucose metabolism in Salmonella typhimurium. A The map of the prophage (Fig. 2) study of a mutant lacking phosphoglucose isomerase. J. 080dhis Biol. Chem. 239:2765-2771. immediately indicates which one of 080dhis 8. Garrick-Silversmith, L., and P. E. Hartman. 1970. Histi- DNA strands must be the "sense" strand for the dine requiring mutants of Escherichia coli K-12. synthesis of his operon messenger ribonucleic Genetics 66:231-244. acid. Since transcription proceeds from the 9. Goldschmidt, E. P., M. S. Cater, T. S. Matney, M. A. and since Butler, and A. Green. 1970. Genetic analysis of the operator towards the structural genes, histidine operon in Escherichia coli K-12. Genetics the chemical orientation of the DNA strand 66:219-229. acting as a template must be from 3' to 5', the 10. Gottesman, S., and J. R. Beckwith. 1969. Directed "R" strand, in which the 3'-5' direction (20) transposition of the arabinose operon: a technique for must the isolation of specialized transducing bacteriophages coincides with the his operon orientation, for any Escherichia coli gene. J. Mol. Biol. 44:117-127. be the "sense" strand for transcription of the his 11. Gratia, J. P. 1964. Resistance a la colicine B chez E. coli. operon. Relations de specificit6 entre colicine B, I et V et phage 662 AVITABILE ET AL. J. BACTERIOL.

T,. Etude genetique. Ann. Inst. Pasteur (Paris) 18. Sato, K. 1970. Genetic map of bacteriophage 080: genes 107:132-151. on the right arm. Virology 40:1067-1069. 12. Kayajanian, G., and A. Campbell. 1966. The relationship 19. Signer, E. R. 1964. Recombination between coliphage between heritable physical and genetic properties of lambda and 080. Virology 22:650-651. selected gal- and gal+ transducing Xdg. Virology 20. Szybalski, W., K. Bovre, M. Fiandt, S. Hayes, Z. 30:482-492. Hradecna, S. Kumar, H. A. Lozeron, H. J. J. Nijkamp, 13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and J. and W. F. Stevens. 1970. Transcriptional units and Randall. 1951. Protein measurement with the Folin their control in Escherichia coli phage A: operons and phenol reagent. J. Biol. Chem. 193:265-275. scriptons. Cold Spring Harbor Symp. Quant. Biol. 14. Martin, R. G. 1963. The first enzyme in histidine biosyn- 35:341-353. thesis: the nature of feedback inhibition by histidine. J. 21. Taylor, A. L., and C. D. Trotter. 1972. Linkage map of Biol. Chem. 238:257-268. Escherichia coli strain K-12. Bacteriol. Rev. 15. Martin, R. G., M A. Berberich, B. N. Ames, W. W. 36:504-524. Davies, R F. Goldberger, and J. D. Yourno. 1971. 22. Vogel, H. J., and D. M. Bonner. 1956. Acetylornithase of Enzymes and intermediates of histidine in Escherichia coli: partial purification and some proper- Salmonella typhimurium, p. 3-44. In H. Tabor and C. ties. J. Biol. Chem. 218:97-106. W. Tabor (ed.), Methods in enzymology, vol. 17b. 23. Yamagishi, H., F. Yoshizako, and K. Sato. 1966. Charac- Academic Press Inc., New York. teristics of DNA extracted from bacterio- 16. Peyru, G., and D. G. Fraenkel. 1968. Genetic mapping of phages t80 and 480pt. Virology 30:29-35. loci for glucose-6-phosphate dehydrogenase, gluconate- 24. Yanofsky, C., and E. S. Lennox. 1959. Transduction and 6-phosphate dehydrogenase and gluconate-6 phosphate recombination studies of linkage relationship among dehydrase. J. Bacteriol. 95:1972-1278. the genes controlling tryptophan synthesis in Esche- 17. Sanderson, K. E. 1972. Linkage map of Salmonella richia coli. Virology 8:425-447. typhimurium, edition IV. Bacteriol. Rev. 36:558-586.