THE IDENTIFICATION OF COMPLEX GENOTYPES IN

Transcription

1 THE IDENTIFICTION OF COMPLEX GENOTYPES IN BCTERIOPHGE T4. I: METHODS. H. DOERMNN ND LINDE BOEHNER Department of Genetics, University of Washington, Seattle, Washington Revision received ugust 17, 1970 HE simultaneous use of more than three genetic markers in bacteriophage Tcrosses is infrequent, and where four or more have been used in a single cross, usually only one of the numerous progeny genotypes has been identified as a unique class among the complex mixture of genotypes. Foss and STHL (1963) have made use of a 4-factor cross in which all 16 segregant genotypes could be identified. More frequently, however, selective tests have been used to score one identifiable type, as, for example, r+ in a complex progeny containing multiple rzz markers (CHSE and DOERMNN 1958). While that type of investigation has yielded much important information, the lack of methods for identification of a larger number of genotypes in a single progeny has severely limited the ways in which multi-factor crosses can be used. The present paper describes techniques by which that handicap can be overcome. The procedures are based on the same considerations as those underlying the replica plating procedure employed by CHSE and DOERMNN (1958) for isolating multiple rzz strains of T4. They have the advantage, however, that the uncertainties present in the earlier method of replicating with velvet from la top layer of soft agar are eliminated. Moreover, any plaque can be used in countless individual tests, thus making possible the identification of many markers in the single phage particle which originated the plaque. This method has been used successfully in our laboratory for simultaneous identification of an acriflavineresistant mutant, a host-range type, and seven rzz markers. It has also been used to identify the ubiquitous amber mutants discovered by &STEIN (see EPSTEIN et al. 1963) and presumably could be applied to the temperature-sensitive mutants of EDGR and LIELUSIS (1964). In general, it seems that a variation of the procedure can be developed to identify any marker for which a selective test is available and whose reversion index and/or transmission coefficient are not too high. MTERILS ND METHODS Bacterial strains: The properties of several Escherichia coli strains used are crucial for distinguishing the various phage alleles. Two are used for making plaques of the phage particles to be tested. The strain S/6 is used whenever amber mutants are not included in any of the genotypes under scrutiny because r and tu mutants produce distinct plaque morphologies on that plating IThis investigation was supported by Public Health Service Research grant GM Much of this work was carried out in the Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee, where it was supported by Public Health Service Research grant C Genetics 66: November, 1970.

2 41 8. H. DOERMNN ND L. BOEHNER host (DOERMNN and HILL 1953). The ambers (am), a class of mutant which will not grow on S/6 or E. coli B (EPSTEIN et al. 1963), are plated on CR-63. On CR-63, as in all strains of the -sensitive K type, T4rII mutants display a wild-type plaque morphology rather than the r type shown on B or S/6. Even though the r type plaque morphology of rll phages is not observed on this host, the testing methods presented here allow identification of the rll mutations present in the phage genome. In making the various kinds of test plates, several streptomycin-resistant bacteria are needed. Strains B/s and K-I2(X)/s have been described by CHSE and DOERMNN (1958). When amber mutations are included in the genomes, K-l2(h)/s must be replaced by K112-12(Xh)#3/s. streptomycin-resistant variant of E. coli strain BR-16 (originally obtained from Professor C. BRESCH) is also useful. The distinctive property of BR-I6 for the present experiments, at least, is that while it is infected and killed by either h42 or am mutants of T4, it prduces phages of neither type unless a complementing strain is also present in the same cell. Table 1 summarizes the bacterial strains used and their properties which are pertinent to the present tests. Phages: ll phages used in the methods described here are mutants of T4D, but T4B mutants can be used in a similar manner. number of rll mutants from various sources have been used, principally those mapped first by EDGR (1958) and EDGR et al. (1962). n important factor for obtaining clear-cut tests for any particular mutant is that its reversion index should not be too high. For example, difficulties are encountered with rll tests of mutants whose reversion index exceeds 5 x Other mutants which can be satisfactorily identified in the manner to be described are ac41 and 941, both of which will grow in the presence of concentrations of acriflavine (or other acridine dyes) which are not tolerated by the wild-type T4. These mutants were obtained from Dr. R. S. EDGR. The former has been described by EDGR and EPSTEIN (1961) and the latter by PIECHOWSKI and SUSMN (1967). The host-range mutant, h42 (EDGR 1958), is also well suited to the testing method because of the availability of the bacterial strain TBLE 1 Bacterial strains used Properties related to identification Bacterial strain Symbol of T4 genotypes CR-63 (orcr-63/1) CR Permissive host for all genetic types. S/6 CR-63/s B/s K-12( h)/s* K112-12(hh) #3/s BR-l6/s S/6 Gives good plaque morphology. Restrictive for am mutants. CR/s B/s Like CR, but streptomycin resistant. Streptomycin resistant. Restrictive for am mutants. K (1)/s Streptomycin resistant. Restrictive for both rll and am mutants. K(Xh)/s BR/s Streptomycin resistant. Restrictive for rll, permissive for am mutants. Streptomycin resistant. Restrictive for h42 and am mutants. Reference EPSTEIN et al DOERMNN and HILL 1953 Isolated from CR-63 CHSE and DOERMNN 1958 CHSE and DOERMNN 1958 Isolated from K112-12(Xh) #3 (WOLLMN 1953) Isolated from BR-I6 which was obtained from C. BRFSCH * Preferred for rll tests when genotypes contain no am mutants.

3 IDENTIFICTION OF GENOTYPES IN PHGE T BR-16. Finally, the am mutant series developed by EPSTEIN is a particularly important group because of its widespread distribution in the T4 genome. From this large group, mutants with low reversion indices were selected for determining the general applicability of the testing method to this category of mutant. General methods: Except for the differences noted below, the media and procedures outlined by CHSE and DOERMNN (1958) were followed here. One difference was that the recipe for bottom-layer agar was modified by altering the sodium citrate concentration. For S/6 platings, plaque morphology seemed optimum with 1.1 grams of sodium citrate dihydrate per liter of medium. When tu and tu+ plaques are to be distinguished, however, in platings on CR or K bacteria, citrate-rich (6.25 grams of sodium citrate dihydrate per liter) plate agar may be used. Plaque mwphology was best when S/6 plates were incubated at 34 C and CR-63 and K plates were incubated at 37 C for hr. second deviation from the techniques of CHSE and DOERMNN was that all platings from which plaques were to be tested were made with exponentially growing cultures of plating bacteria prepared according to the formula of EPSTEIN (1958). These will henceforth be called plaque plates to distinguish them from test plates onto which samples from the plaque contents are transferred. The test plates are made with overnight aerated cultures. For making plaque plates it has been our experience that the closed brewer s type covers made of aluminum with an absorbent liner yielded more reliable results than disposable plastic plates or plates with glass covers. The latter plates, even when incubated overnight before use, frequently showed condensed moisture around the edges. Occasionally the moisture ran over the plate, presumably mixing phage from various kinds of plaques. ny particular plaque tested would, therefore, contain an element of uncertsinty. With the aluminum covers, plates could be used as som as the medium had solidified, and the danger from moisture runs was largely eliminated. Test plates are poured in glass-top plates. They, too, are used as soon as the medium has solidified, but moisture condensed on the covers is wiped out. The reason glass covers are necessary here is that test plates are ospen to the atmosphere for an appreciable interval while tests are being made, and they tend to dry out during that time. When aluminum covers are used for test plates, the additional loss of water through the absorbent disk during incubation is excessive and makes phage growth so sparse that tests become dubious. ll the various kinds of test plates contain 4&50 ml of plate agar per 100 mm diameter plate or ml per 150 mm diameter plate. PPLICTIONS Method Z: Testing individually with glass rods Basic test procedure (described for rll tests): In order to find out whether a certain phage particle carries a particular rzz mutant site, a method is used which, in principle, is similar to the BENZER (1957) spot test. If it is desired to know whether a phage particle from the progeny of a multi-rzz cross contains, for example, r70 in its genotype, it is first plated on an exponential culture of S/6 (or CR-63) and incubated hr. t least one hr before the test is to be made, a 2 ml top-layer of soft agar is deposited on a freshly poured test plate (i.e., poured within the last 10 hr). The top layer contains the following ingredients: (1) 1.5 x los r70 phage particles; (2) 0.15 ml of a mixture of overnight aerated cultures (30 C) of K(x)/s and B/s in a ratio of 50: l by volume; and (3) 2 drops of streptomycin sulfate solution which contains 5 mg per ml. The top layer is allowed to harden for 30 min at room temperature, and the plate is then placed in the refrigerator where it is stored until it can be used. Such plates are usable for at least 8 hr if kept at 2 to 8 C.

4 420. H. DOERMNN ND L. BOEHNER FIGURE 1.-n r70 test plate using Method I. 1.5 X. The bacterial strain K(h)/s was used and the plate incubated 24 hr. The 5 columns represent 5 genotypes of which 5 plaques each we:e tested. The genotypes tested are, from left to right, r73, r77, r70, wild type, and rx4 r77 (r73 is a mutation in the rlzb cistron, and the others are all mutations in rzz). The test plate itself was used as a negative by inserting it into a photographic enlarger and making the print directly. The dark spots therefore indicate areas of clearings. phage is tested by stabbing its plaque with a sterile glass rod and transferring to the test plate by stabbing the latter in some predetermined pattern. In our experience nine stabs in a 5 mm square gives an unambiguous test. The tip of the glass rod will carry phage even to the ninth stab without reinserting it into the original plaque, which thus remains uncontaminated. It is possible to stab the same plaque again and again with fresh glass rods to make additional tests on other types of test plates. In this way, a large number of tests can be made from a single plaque, and the complete genotype of the phage which produced it can be determined. The glass rods used in these tests are made from a 3 mm Pyrex rod,

5 IDENTIFICTION OF GENOTYPES IN PHGE T4 421 drawn to a point, broken off, and fire-polished so that the rounded tip has a diameter of 0.5 to 1.0 mm. It is advisable to leave the test plates uncovered no more than 40 min and no less than 20 min during the testing procedure. Local temperature and humidity conditions may, of course, alter these limits. fter incubation overnight at 37"C, those spots which originated from plaques containing 7-70 phage d l be completely turbid, while those which contain r70+ phage will give clear areas (see Figure 1). further distinction can also be made in the rzz test plates which is sometimes useful. test plate seeded with a phage which contains an rzz (BENZER 1957) marker will show a completely clear spot for any plaque tested which contains no rzz marker. This is presumably because phage growth can begin immediately in all mixedly infected cells. If, however, the phage being tested contains an rzz marker at a different site from the r in the test-plate seeding, a more speckled clearing will result since phage reproduction in K(h)/s cannot proceed until they are infected by an r+ recombinant emerging from the permissive B/s cells. This difference may be seen in Figure 1. To decide whether other non-rzz sites are wild type or mutant, similar tests are made, except that the ingredients of the test plates are different, The composition of the test plate for various other types of markers is given in Table 2. Under some conditions it is useful to incubate test plates as long as 48 hr. This is generally not necessary unless closely linked markers in a single cistron are involved. The use of copy p2ates: s the number of tests to be made from a single plaque becomes larger, two types of problem develop: First, the plaques tend to dry out while uncovered during the prolonged period required for many tests from a single plate. Secondly, the smallest plaques in particular become so macerated from many stabs that it is not always certain that the later stabs are made in the phage-rich area of the original plaque. Both of these problems can be averted by use of copy plates. fter hr incubation of the plaques which are to be tested, they are stabbed with glass rods and copied on fresh plates seeded with either S/6 or CR-63. Glass-top plates are used for this purpose and are left uncovered for a min interval similar to test plates. It has proved useful to make, for each plaque being copied, several stabs close together on the copy plate. This gives a continuous clearing for even the smallest plaques copied. fter hr of incubation, tests are made from the copies. Such plates stored in the refrigerator for as long as a week give dependable tests, even with difficult marker combinations. Errors when rare multiple recombinations are required during incubation of the test plate: The method, even in the first trials yielded completely unambiguous results when used in testing single-r plaques against single-r test plates. It was, however, necessary to find out whether the multiple recombinations which in some cases are required to make r+ particles capable of growth in h-lysogenic bacteria on the test plate, would be so rare that a clear spot might fail to develop. This would, of course, result in an incorrect decision as to that particular locus in the phage particle being tested. Since the phage particle seeded onto the test plate is always a single r, the highest order of multiple recombination needed to produce a totally r+ phage will be a double crossover. low frequency of double

6 422. H. DOERMNN ND L. BOEHNER TBLE 2 Conditions used in making tests for specific mutants of T4D' Titer of phage Quality Mutant+ suspension Bacterial mixture$ of tests amb23 amb16 amnl32 amel8 ad76 ame389 ams71 amb6 amn85 ams6 amn54 ad453 amb25 amn58 am455 ad265 amn52 amn82 amb256 amn135 ame609 amn53 amb17 ad52 ams29 amn122 amb22 amc42 ame1 7 amn93 amni28 amn69 amb251 amn5o amn98 ame1236 amb258 ame355 ame51 ams2 amn81 amn130 amb2o ame509 ame9oo 5 x 108 ex x x B/s : 1 CR/s 10B/s : 1 CR/s 20B/s : 1 CR/s 10 B/s : 1 CR/S 20B/s : 1 CR/s 50 B/s : 1 CR/s 20 B/s : 1 CR/s B B

7 IDENTIFICTION OF GENOTYPES IN PHGE T4 423 am8-82 amh26 amb255 ad66 amn131 ams105 ams92 ad60 amb252 amh17 rll rll rb41 rb41 ac41 h42 (ZYS) (ZYS) (10) (16) (26) (26) (27) (27) (35) (52) (absence of am mutants) (presence of am mutants) (absence of am mutants) (presence of am mutants) Titer of phage Quality sumension Bacterial mixturet of tests 5 x x 107 2x x 107 I x x x x x x 10s 50 B/s : 1 CR/s B/s only 20 B/s : 1 CR/s 50 B/s : 1 CR/s 3 B/s : 1 CR/s 5 B/s : 1 CR/s 5 B/s : 1 CR/s 3 B/s : 1 CR/s 10B/s : 1 CR/s 50B/s : 1 CR/s 1.5 x K(h)/s : 1 B/s,C 1.5 x K(hh)/s : 1 CR/s B,C 1.0 x K(X)/s : 1 B/s B B B B B,C 5.0 x 10s 8 K(h)/s : 1 CR/s B,C 2.0pg acriflavine per ml bottom layer agar with CR in top layer (useful only in absence of am mutants) BR/s bacteria used * ll Method-I tests are made on a 2 ml top layer of soft agar which is seeded with 0.1 ml cf phage suspension (where appropriate) and 0.15 ml of bacterial mixture. Except for ac41, 0.5 mg of streptomycin sulfate is added to the top layer of each test plate. For Method I1 (described later) all components of the top layer are increased by a factor of Most of the am mutants were obtained from R. S. EDGR and R. H. EPSTEIN. The numbers in parentheses are the gene numbers assigned by EDGR, DENHRDT and EPSTEIN (1964). ll mutants with the letter S preceding the mutation number were isolated in the present program from phage grown in the presence of bromodeoxyuridine. 2 Volumetric ratio of overnight aerated cultures $ ll tests listed are satisfactory when no other mutant in the same cistron is to be distinguished. Quality symbols give the following additional information: = Good tests even with other mutants in the same cistron B = Tests difficult although possible to distinguish when another mutant is included in the same cistron C = Improved and generally satisfactory with hour incubation of test plates. crossovers is anticipated when the phage being tested carries r77+ surrounded by the closely linked markers r70 on one side and rb42 on the other, and when the test-plate is seeded with 7-77 (for map, see DOERMNN and BOEHNER 1963). Of these markers, rb42 is located in the rzzb cistron while the other 2 are located in the rzz cistron. It is possible, therefore, that functionally complementary combinations of phages might arise following single crossovers and these might facilitate the appearance of I+ phages and clear spots on the test plates. more restrictive combination of 3 markers would perhaps be one where all 3 markers are in the same cistron. as, for example, r64,r70, and r77. For each of the 2 sets of 3 markers, 200 plaques doubly mutant with the outside markers (i.e., r70 rb42 in the former case and r in the latter) were tested on plates seeded with phage carrying the centrally located r marker (r77 in the former case and r70 in

8 424. H. DOERMNN ND L. BOEHNER the latter). n example of such tests is shown in Figure 1. ll tests were conclusive in that easily recognizable clearings were found for each plaque tested. Closer combinations of markers would, of course, necessitate new controls. Similar tests with am mutants using condkions given in Table 2 yielded satisfactory results. It seems, however, to be more difficult to resolve three amber mutations in a single cistron than it does to distinguish three or more rzz or rzzb mutations. Errors due to false positive tests resulting from reverse mutations during plaque growth: Genetic reversions might give rise to a second source of error. If, for example, reversions occur frequently during growth of an rzz plaque, the revertants would make a clear spot, implying an r+ genotype at the site being tested. The use of copy plates, with an additional cycle of growth, would probably tend to accumulate revertants and perhaps exaggerate this source of error. Six rzz and 15 am mutants were checked for the occurrence of such false positives when tests are made with Method I. Stocks of each were plated against CR- 63, and a set of 234 plaques of each copied on CR-63. Tests of the copied plaques were made by the standard procedure, seeding the test plates with the homologous mutant. Of the rzz s, three (r61, r64, and r73) have no detectable reversion frequency, and, as expected, no errors were found in the 702 plaques tested. Stocks of the other three (r70, r77, and rb32) always contain revertants in low frequency. However, only one test (among the rb42 set) was found to be misleading out of the 702 tests made. It seems reasonable to ignore any error from this source when these particular rzz mutants are used. Each of 15 am mutants investigated similarly showed reversions in stock suspensions. Seven (amb16, amn52, amn54, amn58, amn82, amn85, and ams71) yielded no test error, and four (amb6, ams6, ams52, and amslo5) gave one doubtful test each. Three others (amn131, ams29, and ams60) produced one positive test each. The remaining mutant (ams92) yielded three false positives in the 234 plaques tested. The frequency of misclassification of these ambers due to revertants seems negligible, with the possible exception of ad92 tests. It should be called to attention here that any phage which is heterozygous at a given site segregates the wild type during the growth of its plaque. Heterozygous sites are therefore misclassified as wild type both in Method I and in Method 11. For measurement of large recombination values this error can be neglected, but when closely linked markers are being studied the proportionate error becomes large. more detailed and quantitative discussion of this complication is reserved for a subsequent report. Method ZI: Testing large numbers with a replicating technique Replicating test procedure: Method I is practically feasible as long as the number of markers per cross is not too large. It has been extensively used in 7-factor crosses by DOERMNN and BOEHNER (1963) and in a 9-factor cross by WOMCK (1963). For experiments with larger numbers of markers, a more efficient method is needed and a replicating procedure was consequently devised. While (the

9 IDENTIFICTION OF GENOTYPES IN PHGE T4 425 principle of Method I1 is the same as for Method I, the modifications require that it be described and tested separately. The velvet replicators used by CHSE and DOERMNN (1958) are not dependable enough for $he present type of experiment because they tend to pick up large areas of the soft-agar #top-layer, making repeated replications from one master plate impossible. Metal replicators were therefore constructed using No. 18 gauge spring-temper stainless steel wire mounted in a babbict metal base, leaving a uniform length of about 1.27 cm protruding. The wires were originally spaced 4.33 to the cm in rows cm apart. This high density of inoculating wires prevented complete burning away of the alcohol used to sterilize the replicators, so that it was necessary to remove every even-numbered wire in alternate rows. Copy plates are essential intermediates when using replicators of this type, especially when crosses include numerous amber mutants which bring about considerable plaque-size variation. In order to make the number of tests per replication as large as practical, Petri dishes of 150 mm diameter are used for copy and test plates. Both are prepared as in Method I, except that the soft-agar layer is increased to 5 in1 and the other components of the test (bacteria, indicator phage, streptomycin, etc.) are increased by a factor of 2.5. Replicators are sterilized by standing them for 5 sec or longer in 3-5 mm of 95% ethyl alcohol which is then burned off. little time must be allowed for cooling and it becomes expedient to have a set of 6-12 replicators if many tests are to be made. fter a number of cycles of replication and sterilization it is useful to brush the replicators with a wire brush to remove adhering bits of soft agar which tend to accumulate. Optimum tests appear to be obtained when test plates are not stored in the refrigerator but used directly after the soft agar has hardened. The lids are removed 5-10 min ahead of the replicating process to allow evaporation of excess moisture. It is advisable to finish replication as soon as possible after pouring the soft agar on the test plates, but even when delayed as long as three hours, satisfactory tests are generally experienced. If it becomes necessary to refrigerate the test plates, they should be removed from the refrigerator and the lids removed for min ahead of the replicating process. fter replicating they are left open min longer, depending on atmospheric conditions. Figure 2 shows a section of a copy plate and 4 test plates made by Method 11. Controls against misidentifications: The replicating procedure was checked for the same two sources of error as were investigated with Method I. Plaques of the double mutants r70 rb42 and r64 r77 were copied and replicated to test plates containing r77 and r70, respectively. In the total of 700 tests none failed to show a definite clear spot, indicating that under proper test conditions the double crossover is frequent enough to permit recognition of the centrally located r+ with a very high degree of reliability. The possibility of error arising from reverse mutations which could give false positive tests was also checked for many of the mutants. The procedure employed was like that used for Method I, except that copy plates were replicated. The tests carried out are listed in Table 3. The frequencies of errors were not high enough

10 COPY PLTE PHGE COPIED am am am am WILD TYPE N54 S52 S60 S92 5M IOM 8M I I I I I I I am N 54 TEST am S52 TEST ams60 TEST am S9i TEST FIGURE 2.-Sections through n copy plate and 4 Method I1 test plates replicated from it 1.2 x. Two plaques of 8 different genotypes were copied and tested. The designation 5M indicates phage carrying the 5 mutant genes amn85, ams71, ams60, ams92, and amslo5. Phage 10M carries r61, r64, r70, r77, rh42, amn52, ams6, ams60, ambl6, and amn82. Phage 8M carries amn58, amn54, r67, amn85, ams92, ams29, amsio5, and ams52. The mutations ams6o and ams92 are both in gene 27. s in Figure 1, the copy and test plates themselves were used as photographic negatives.

11 ~ IDENTIFICTION OF GENOTYPES IN PHGE T4 TBLE Frequency of false positive tests in mutant identification by Method I1 Three hundred forty-fiue plaques from phages carrying a single mutant were copied on CR-63 and replicated to test plates seeded with the homologous phage ~~ List of mutants tested Errors' in 345 tests amnl35 ad16 amb23 amn53 amb17 ame355 ams52 amni31 ad105 ams6o amb6 amn85 ams6 amn54 amn58 amn52 r73 rb45 rb41 red220 r70 r71 r64 r61 red219 amni30 0 amb256 ad92 ad71 rb42 amn81 amn82 1 amb251 ams29 ac41 2 *Whenever doubt existed about how a test should be scored, it was classified as an error. dditional evidence exists which indicates that when tests are made on plaques from multimutant phages, false positives are even less frequent than indicated by tests of single mutants. to cause concern, particularly in view of the fact that tests from multiple-mutant stocks (which are generally involved in crosses which employ replication) show a lower frequency of false positives than do the single-mutant tests on which the table is based. Multi-amber phages often make very small plaques which may give rather sparsely cleared areas on the copy plate. To obtain dependable tests it is sometime? necessary to recopy the original copy in order to produce a more or less completely cleared area. This raised the question whether repeated copying accumulates revertants which might give misleading tests. s a check against this possibility plaques from a phage stock marked with one rzz and 21 am mutations were copied and tested for each marker. (The markers were r219, amb256, amn135, amb251, ambl6, amb23, amb17, ams52, ams105, ams29, ams92, ams71,amb6, amn85, amn54, amb25, am455, amn52, ams2, amn81, amn82, and amnl30.) fter the first test, the copy spots were recopied on a fresh plate and again tested for each marker. mong 210 plaques handled in that way, none showed a nonmutant test for any of the markers in the test from the original copy. The tests of the second copy plate showed one nonmutant test (for amb251). When 140 of the second copies were again recopied, four reversions (all amb251) were found. Thus, for the markers tested, a single recopying of small plaques does not appear to be hazardous. The metal replicators used for Method I1 were constructed by Mr. BILEY F. MOORE of the Vanderbilt University Medical Center Machine Shop. The authors wish to acknowledge his extensive help and cooperative spirit in designing the replicators as well as constructing them.

12 H. DOERMNN ND L. BOEHNER SUMMRY Two methods have been developed for determining the genotypes of individual phages derived from complex crosses. Method 11, which makes use of replicators especially designed for use with soft-agar overlays, has two main advantages: (1) when many markers are being tested in one phage, the process becomes much more efficient when replication is employed; (2) the orientation of the plaques is the same on all test plates, thus eliminating accidental transpositions and other sorts of technical errors. On the other hand, Method I is preferred when the number of tests per phage is only 2 or 3 because it is then unnecessary to interpolate a copy plate between the original plaque and the final test. Both methods seem to yield equally reliable results when mutants with low reversion indices are selected and if the number of markers in one cistron is not too great. LITERTURE CITED BENZER, S., 1957 The elementary units of heredity. pp In: The Chemical Basis of Heredity. Edited by W. D. MCELROY and B. GLSS. Johns Hopkins Press, Baltimore. CHSE, M. C. and. H. DOERMNN, 1958 High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43 : 33S-353. DOERMNN,. H. and L. BOEHNER, 1963 n experimental analysis of bacteriophage T4 heterozygotes. I. Mottled plaques from crosses involving six rll loci. Virology 21 : DOERMNN,. H. and M. B. HILL, 1953 Genetic structure of bacteriophage T4 as described by recombination studies of factors influencing plaque morphology. Genetics 38: 79L90. EDGR, R. S., 1958 Mapping experiments with rll and h mutants of bacteriophage T4D. Virology 6: EDGR, R. S., G. H. DENHRDT and R. H. EPSTEIN, 1964 comparative genetic study of conditional lethal mutations of bacteriophage T4D. Genetics 49 : EDGR, R. S. and R. H. EPSTEIN, 1961 Inactivation by ultraviolet light of an acridine-sensitive gene function in phage T4D. Science 134: EDGR, R. S., R. P. FEYNMN, S. KLEIN, I. LIELUSIS and C. M. STEINBERG, 1962 Mapping experiments with r mutants of bacteriophage T4D. Genetics 47: EDGR. R. S. and I. LIELUSIS, 1964 Temperature-sensitive mutants of bacteriophage T4D: Their isolation and genetic characterization. Genetics 49 : EPSTEIN, R. H., 1958 study of multiplicity-reactivation in the bacteriophage T4. I. Genetic and functional analysis of T4D-K12(X) complexes. Virology 6: EPSTEIN, R. H... BOLLE, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE L TOUR, R. CHEVLLY, R. S. EDGR, M. SUSMN, G. H. DENHRDT and I. LIELUSIS, 1963 Physiological studies of conditional lethal mutations of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28: Foss, H. M. and F. W. STHL, 1963 Circularity of the genetic map of bacteriophage T4. Genetics 48: PIECHOWSKI, M. M. and M. SUSMN, WOLLMN, E. L., cridine-resistance in phage T4D. Genetics 56: Sur le determinisme ggn6tique de la lysogenie. nn. Inst. Pasteur 84: WOMCK, F. C., 1963 n analysis of single-burst progeny of bacteria singly infected with a bacteriophage heterozygote. Virology 21 :