CO-TRANSCRIBED CISTRONS IN BACTERIOPHAGE T4*

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1 CO-TRANSCRIBED CISTRONS IN BACTERIOPHAGE T4* FRANKLIN W. STAHL, JEAN M. CRASEMANN, GHARLES YEGIAN, MARY M. STAHL AND ATSUO NAKATAZ Institute of Molecular Biology, Uniuersiiy of Oregon, Eugene, Oregon Received September 2, 1969 PAIRS of co-transcribed cistrons have been identified in T4 by the analysis of cis-trans position effects between amber mutants (STAHL and MURRAY 1966; STAHL et a1 1966; NAKATA and STAHL 1967). This communication is our final report of the results of a survey of the T4 genome for such intercistronic position effects and of the analysis of each discovered case. The finality of this report implies only that we are through looking, not that there is nothing more to find. Our survey and analysis were composed of the following elements: (1) A systematic search for cis-trans position effects between amber mutants in neighboring and near neighboring genes in the late region of T4; (2) The determination of the direction of transcription of the discovered co-transcribed pairs relative to the conventional T4 linkage map; (3) The determination of the extent of the (6 transcriptons detected using a sensitive modification of the cis-trans test. A 6 transcripton is a group of co-transcribed cistrons as defined by the cis-trans position effect. Operons (JACOB and MONOD 1961) turn out to be transcriptons, but transcriptons need not be operons. That is, we have no evidence that the rates of expression of the members of a T4 transcripton are coordinate, or are under the control of a macromolecular repressor which acts upon an operator according to the presence or absence of a small-molecule effector ; (4) A spot-check for cis-trans position effects between amber mutants in the early region using the sensitive version of the cis-trans test employed in determining the extent of transcriptons in the late region. This method determined the direction of the polarity effect as it detected its existence; (5) A careful search for cis-trans effects between the A and B cistrons of the rll region. MATERIALS AND METHODS Phage strains: The amber (am) and temperature-sensitive (ts) mutants of T4D employed in this study were isolated by R. S. EDGAR, R. H. EPSTEIN and their colleagues. Some of them came to us via SIDNEY BRENNER; the rest came directly from EDGAR. Mutants in the rll region (of T4B) were ohtained from SEWAIL CHAMPE or SEYMOUR BENZER with the exception of the ts mutants which JAN DRAKE sent us. The actual mutants employed will be indicated in the appropriate parts of RESULTS. Bacterial Strains: Escherichia coli strain CR63 was used for stock-making; strain BB was used * Supparted by National Science Foundation GB 8109, and by Public Health Service 1 PO1 GM NSF Postdoctoral Fellow. Present address: Research Institute for Microbial Diseases, Osaka Univerity, Yamada-kami, Suita, Osaka, Japan. Genetics 64: February 1970.

2 158 F. w. STAHL et al. as the host in all complementation tests except those involving the rll region, for which G(1) was used. Methodology: Complementation tests and controls were performed as described by STAHL et al. (1966) and by STAHL and MURRAY (1966). RESULTS Suruey of the late region of the map: Many of the genes of T4 are involved in steps in the infectious process which follow the onset of DNA synthesis. These genes, which act late, are located primarily in a single long segment of the circular linkage map. For most of the known genes in this segment, we compared phage yields from cis and trans complementation tests between nearest neighbors and neighbors once, twice, or thrice removed (as judged from the T4 map in Figure 1). The ratios, yield from trans test/yield from cis test, are presented in Table 1. An overall impression of the data may be better obtained from Figure 2. We have plotted the number of plaques counted in estimating the yield from the trans FIGURE 1.-Linkage map of T4. All of the genes shown, except e, were employed in the present study. Solid arrows indicate the extent and direction of co-transcribed groups of genes as detected by the present studies (see RESULTS). Broken arrows indicate the direction of transcription of 4 genes established by other workers (STREISINGER et al. (1968) for the e gene; CRICK et al. (1961) and BENZER and CHAMPE (1962) for the riia and B genes; SARABKAI et al. (1964) for gene 23). In this paper, the late region is the set of genes from 53 clockwise to 38.

3 TRANSCRIPTONS IN PHAGE T4 159 TABLE 1 Summary of cis-trans survey of the late region of the T4 map Gene-pair numbers I I f14-i5 fl4-i5 fl4-i Amber mutant numbers H28 H28 NI35 NI35 NI35 NI35 NI17 NI17 NI17 NI17 NI15 NI15 NI15 NI15 NI32 NI32 NI32 NI32 B255 B255 B255 B255 NI28 NI28 N69 N69 B2O* * BU6 BU15 NG251 B20 NI33 NI33 N66 N66 N56 N56 NI35 NI17 NI17 NI15 NI32 NI15 NI32 B255 NI NI28 B255 NI28 N69 B255 NI28 N69 NI28 N69 B20 N69 B20 B20 NI33 N66 N56 NI33 NI33 NI33 NI33 N66 N66 N56 N56 E18 E18 E1137 trans gield cis.yield (2) 0.57 (2) 0.67 (2) 0.82 (2) (2) (2) 0.29* 0.75 (4) W * The wild type used in this cis test was a revertant of. * * These ambers gave no wild-type recombinants in a standard cross with each other.

4 160 F. w. STAHL et al. TABLE I-(continued) Gene-pair numbers Amber mutant numbers irurzs yield cis yield IS IS N56 E18 E372 E18 E372 E18 E1137 E1137 N50 N50 N90 N90 B270 B270 B17 B17 c31 C31 c11 c11 N131 NI31 S29 S29 S29 S29 S29 NI20 NI20 A452 A452 B7 B7 N85 N85 C235 c235 C72 C72 N54 N54 A453 A453 N134 N134 N58 N58 B252 B252 N50 E1137 E1137 N50 N50 N9O N50 N90 N90 B270 B270 B17 B17 c31 C31 C11 c11 N131 Nl31 S29 S29 NI20 NI20 A452 B7 N85 C235 A452 B7 B7 N85 N85 C235 C235 C72 C72 N54 N54 A453 A453 N134 Ni34 N58 N58 B252 B252 E1 E1 N (2) (6)

5 ~ ~~~ ~ ~ ~ TRANSCRIPTONS IN PHAGE T4 161 TABLE I-(continued) El N El B Cis-trans tests as defined by STAHL et al. (1966) were performed with a set of amber mutants from the late reeon of the T4 map. For the most part, the tests involved a random set of ambers-i.e., the ambers we brought to Eugene from the simple set BRENNER acquired from EPSTEIN and EDGAR. However, sxne of the tests involved other mutants. When the choice of those mutants was prejudiced by Considerations in addition to availability, the line is marked +. Where useful, our motives are discussed in the text. A few of the tests not daggered probably should be. Except for the single indicated case (13-15), the tests reported here are standard cis-trans comparisons (see legend to Figure 2). All the standard tests performed in our survey are reported here except for a few cases in which the (trans/cis) yield-ratio significantly exceeded unity. All such examples were ascribable to properties of the double am stock used in the cis test. The values reported here are those obtained with freshly prepared double am stocks. In general, tests weie performed once except where indicated by a numeral in parentheses following entries in the last column. The numeral indicates the number of tests, and the (trans/cis) value reported is the ave:age of the values observed. test us. the number counted in the cis test. In Figure 2, it is apparent that most of the tests gave a slightly smaller trans yield than cis yield. We judge these slight cis-trans effects to be primarily a consequence of the cell-to-cell variability in actual numbers of input phage particles adsorbed. As described in the APPENDIX, this finite-input effect can lead to (trans/cis) ratios as small as 0.7 when the multiplicity of infection is 10. Because of this complicating phenomenon, and because of experimental variability, we decided to ignore any (trans/cis) ratio larger than 0.40, and to arralyze all cases in which that ratio is equal to or less than We found six cases in which nearest-neighbor genes gave a (trans/&) ratio less than 0.4 and one case in which a low ratio was found between neighbors once removed (see Figure 2). Of the six cases involving nearest neighbors, two (51-27; 34-35) had in fact been discovered and described previously ( STAHL et al. 1966). The cis-trans effect for gene pair is related to that for genes 13 and 14 (see below). Thus, our fairly thorough search uncovered four previously undescribed cases of interest. During the course of our survey, J. KING told US that he had found pcor in vitro complementation between ambers in genes 18 and 19. We repeated our cis-trans test twice using our ambers in 18 and 19. The three tests gave (trans/cis) ratios of 0.51, 0.35 and 0.37, respectively. (The last test used a revertant of E18 as wild type.) We then obtained a different amber (E372) in gene 18 from KING. In three cis-trans tests us our aniber in gene 19 we obtained (trans/cis) ratios of 0.24, 0.17 and 0.31, confirming the relevance of KING S observation. (The last two tests used the revertant of E18 as wild type.) The four new cases we discovered plus the 18(E372)-19 pair were then analyzed as follows: (1) For each case, the presumptive direction of transcription (i.e., the direction of the polarity effect) was determined by one or both of the two methods described by STAHL et al. (1966). In one method, the degree of complementation between an am mutant in one cistron and a ts in the other distinguishes direction. The other method varies the relative multiplicity of two complementing ambers. In each case, the polarization was found to be clockwise on the conventional map. (2) A plausible trivial explanation of each cis-trans effect was ruled out. We

6 162 F. w. STAHL et al. e FIGURE 2.-Results D PLAQUES COUNTED IN CIS TEST of cis-trans complementation tests between am mutants in nearby genes in the late region of T4. For a given pair of am mutants, cis and trans tests were performed in parallel on samples from the same exponential culture of the am-restrictive host, BB. In the truns tests, infections were by 5 particles per cell of each of the two single mutants; in the cis tests infections were by 5 particles each of wild type and the relevant double mutant. Following phage adsorption (in cyanide) the two cultures were diluted identically. Following cell lysis, the cultures were again diluted and plated identically. The graph plots the number of plaques arising on the plates from the trans test us the number from the cis test. The data plotted are those from which the (trans/cis) ratios of Table 1 were calculated. In cases where more than one pair of tests was made, the first one performed is graphed here. Tests involving neighboring genes (as defined by the map in Figure 1 ) 0 Tests involving neighbors once removed A Tests involving neighbors twice removed 0 Tests involving neighbors thrice removed As described in the text, the truns values are expected to be less than the cis values in the absence of any position effect. For tests involving two genes whose products relate stoichiometrically to the phage yield and are not produced excessively, the points should fall on the upper diagonal line (see APPENDIX). The lower diagonal separates those tests we chose for further study from those whose deviation from the expected was judged to be uninteresting. The variability in the cis values is due in part to partial dominance of some of the ambers and in part to variability among bacterial cultures.

7 TRANSCRIPTONS IN PHAGE T4 163 selected am+ revertants of each of the apparent polar mutants. Stocks of these revertants were then used as the wild-type parent in a cis-trans test. In each case, the revertant behaved indistinguishably from our standard wild-type stock, ruling out the possibility that the original cis-trans effect was due to an extraneous mutation in the apparently polar member of the pair of amber stocks. To all seven cases we then applied a sensitive variation of the basic cis-trans test to define the distal limit of each of the transcriptons. The basic survey showed that the polar effect of gene 13 extended at least as far as gene 15. In none of the other cases, however, was there any suggestion that the transcripton was more than two genes long. However, in any single case this could have merely reflected one of the limitations in the sensitivity of our standard method of survey. If a gene product is made in excess of the requirements for a normal yield of mature phage (as measured under our conditions), a standard cis-trans test may fail to reveal a reduction in the amount of gene product resulting from the presence of a nearby polarity mutation. We removed this source of insensitivity as follows: we obtained (from EDGAR) a ts mutation in the next-nearest-neighboring gene (when possible) in the clockwise direction from each polar mutant. We then performed cis-trans tests of the following sort: tram test am + ts cis test am + am + + am + + ts The tests were conducted at a temperature sufficiently high to reduce the yield in the cis test to about 5-50% of that observed in the test involving the same am mutants without the ts mutation. The results of these tests are summarized in Table 2. They permit the following conclusions: (a) The transcripton, 53-5, probably does not include gene 6. (b) The transcripton, 9-10, does include gene 11 but probably does not include gene 12. (c) The transcripton, , probably does not include gene 17 (no ts available in gene 16). (d) The transcripton, , includes genes 28 and 29 (no ts available in 48 or 54). (e) The transcripton, 48+54, probably does not include gene 30. (f) The transcripton, 34-35, probably does not include gene 36. Spot-check of early genes for polarity mutants: The genes which play essential roles early in the T4 life cycle are segregated (with a few exceptions) into one arc of the map. Many of these genes have been shown to code for proteins whose role is catalytic. A modest reduction in yield of one of these proteins resulting from a polarity effect of a nearby am mutation would probably not be reflected in a reduction in phage yield under our conditions (cultures chloroformed 70 minutes after diluting out cyanide). Therefore, we applied to two pairs of adjacent early genes the sensitive test which we used in the late region

8 164 F. w. STAHL et al. TABLE 2 Determination of the distal limits of transcriptions in the late region Standard Tests Sensitive Tests Geie-pair numbers Temp. I-Y C ( ts, = A25) 31 am, X amy irms test wild X am,amy cis test amstsy X am,, trans test (4) Is,, X am,am,, cis test (4) (ts, = N6) (4) (4) (ts, = B60) (4) 4-5 (4) (4) 49 (4) (tsy = A7) (40) (4) ( tsy = L2) (4) 35 (4) (4) 64 (4) (fs, = A23) (IO) 64 (IO) (ts, = A61) (4) 27 (4) 100 (4) 84 (4) (ts, = Ll03) (12) (8) ( ts, = BZO) 3) (4) 41 (4) (4) 65 (4) (ts, = N41) (5) (5) Standard cis-trans tests and sensitive tests were performed at selected temperatures on samples of a single bacterial culture. The amber mutants employed were those used in the survey (Table 1). The ts mutants employed are indicated with each set of tests. Yield is in units of numbers of plaques counted except when an entry is followed by a number in parentheses. In those cases, the entry indicates the yield relative to the other tests. The actual number of plaques counted is equal to the value of the entry multiplied by the number in parentheses. to determine transcripton length. Of course, these tests suffer from the weakness that we have no knowledge as to whether the am mutants chosen are sufficiently proximal in the transcripton (see NAKATA and STAHL 1967) to manifest polarity. Thz results (Table 3) gave no evidence of polarity of gene 42 on 43,43 on 42 or 44 on 45. However, an amber in 45 does exert a polar effect on gene 44. The nearest available ts mutant distal to gene 44 was in gene 43. Application of the sensitive cis-trans test between genes 45 and 43 showed that the transcripton probably does not extend to gene 43.

9 TRANSCRIPTONS IN PHAGE T4 TABLE 3 Sensitiue cis-trans tests between adjacent early genes 165 Question Test am4,amqs x wild 42 polar on 43? am42am42 X ts43 am4ets4s x a37243 am4,amq3 x wild 43 polar on 42? am42am4s ts40 ts42 am43 Yield at indicated temwrature ~ 28OC 40 C (5) (5) 28 C 35oc 36 C (4) 112 (4) (4) 74 (4) 28OC 37 C 38OC (2) 113 (10) (2) 62 (IO) 28 C (4) 33 (4) Four spot-checks were performed for polarity in the early region. The mutants employed were am C87 and ts L23 in gene 42, am C228 and ts L92 in gene 43, am E20 and ts L259 in gene 45 and ts B22O in gene 44. The ambers used in gene 44 were N82 in the test of 44 polarity on 45 and E2057 in the reverse test. Yield is in units of numbers of plaques counted except when an entry is followed by a number in parentheses. In those cases, the entry indicates the yield relative to the other tests. The actual number of plaques counted is equal to the value of the entry multiplied by the number in parentheses. Tests involving the A and B cistrons of the rii region: CRICK, BARNETT, BRENNER and WATTS-TOBIN (1961) and BENZER and CHAMPE (1962) showed that the A and B cistrons are transcribed counterclockwise, from A to B. We sought evidence that they are co-transcribed by the sort of sensitive cis-trans tests shown in Figure 3. The results of these tests are summarized in Figure 4 and Table 4. They show that even those A cistron nonsense mutants farthest from the B cistron have at most a small polarity effect on that cistron. This conclusion has been reached independently by MCCLAIN and CHAMPE (personal communication) who made chromatographic measurements of B cistron peptide in the presence of nonsense and frame-shift mutations in the A cistron. The significance of this result with respect to the possibility of co-transcription of the two cistrons in the rll region will be confronted in the DISCUSSION. DISCUSSION Shortcomings of the survey and analyses: Our survey and analyses have several shortcomings; in addition to random variability, we can identify the following: 1 ) Some transcriptons may be overlooked because the relevant amber mutant we used in our survey was not a strong polar mutant. Two observations give us a

10 166 cis test F. w. STAHL et al. trans test X X t,s del - rlib FIGURE 3.-Polarity in the rll region from the A to the B cistron was sought via cis-trans tests of the sort diagrammed. NO^" is anyone of the following ambers or ochres in the A cistron: nonsense mutant N55 NB4777 HBlI8 AI - A2 A2 ~ left end of A cistron HBIPY HB84 1 right end of N21 The deletion in the B cistron is WS.33. The ts mutants in the B cistron were T54 or 515. Complementation tests were conducted at several temperatures some of which were high enough to reduce B cistron activity to the point where it limited the phage yield. The tests were conducted on the nonpermissive (a-) lambda lysogen G(A). TABLE 4 Sensitive cis-trans tests in the rii region as diagrammed in Figure 3 rlla non och N55 och N55 am NB4777 am NB4777 amhbl18 amhb129 am HB84 och N21 och N21 rlib ts 51 5 T T54 T54 T T54 Yield/infected cell trans CIS Tests were carried out in G(A) at 35 C. The locations of the nonsense mutants are given in Figure 3.

11 TRANSCRIPTONS IN PHAGE T4 2oo I I Control I Temperature ( C 1 FIGURE 4.-Complementation tests between A cistron and B cistron rll mutants under conditions in which the amount of B product limits the phage yield. The tests, performed in G(h) at the indicated temperatures, were as follows: r11a4amnb4777 riibts515 X rllbdelw8.33 (trans), riiaamnb4777 riibdelw8.33 x rriibts515(cis), and riiaamnb4777 riibts515 X riiaamnb4777 riibdelw8.33 ( leaking control). rough picture of the magnitude of this limitation. (a) NAKATA and STAHL (1967) showed that 6 out of 8 ambers in gene 34 were about equal in their polarity effect on gene 35 while the two ambers closest to gene 35 were weaker. (b) Our original amber in gene 18 did not manifest polarity on gene 19 (on its first test) though KING had encountered an amber which did so (see RESULTS). 2) The distal member of a pair of genes being tested may normally produce in excess of the requirements for a normal yield of phage. Two observations within the present work bear on this possibility. (a) The (trans/cis) ratios in standard tests in the late region tend to cluster about 0.7 (Figure 2). This is the value calculated (in the absence of any position effect) if both genes are producing in amounts which limit the phage yield. We recognize a circular element in this reasoning which is somewhat alleviated by numerous casual observations that, for genes in the late region, mixed infections by am and wild-type phage (in strain BB) give smaller yields than do infections by wild type alone. (b) The sensitive cis-trans tests did show that some late transcriptons are longer than they

12 168 F. w. STAHL et al. appeared to be from the results of the standard tests. From these considerations, which point in opposite directions, we are unable to make any assessment of the seriousness of this shortcoming in our analysis. 3) The set of mutants with which we worked did not represent all the presently known genes of T4. Furthermore, the probable existence of as yet undiscovered late genes contributes as well to the incompleteness of our search, If most transcriptons of T4 are short, as they appear to be, then many would be missed in a search which failed to involve most of the genes. 4) We observed several cases in which the (trans/&) ratio was conspicuously (and reproducibly) greater than unity. In each case, this outlandish result disappearzd when we used a freshly prepared double am stock in the cis test. Perhaps a few (trans/&) ratios which were greater than 0.4 (but equal to or less than unity) would have been less than 0.4 had we prepared different stocks for the tests. 5) In each trans test, wild-type transciptons arise by genetic recombination. The yield of product from a distal member of a recombined transcripton is no longer subject to depression by a polarity effect of the amber in the proximal gene. If the transcripton in question does ordinarily contribute function after the onset of recombination, then position effects within it will be diminished. The seriousness of this limitation in our methods can be assessed by studies on expression of alleles arising by recombination between members of a pair of intragenic markers. SBCHAUD and STREISINGER ( 1962) demonstrated that recombinant-type, host-range protein (tail fiber) is in fact provided in cells mixedly infected by two strains carrying ts markers in the host-range gene. Experiments more directly applicable to our present problem have been performed by STAHL and STAHL (unpublished). In these experiments, nonpermissive (BB) cells were infected by a few particles of each of two strains bearing recombinable am mutations in the same gene. Phage yields were obtained which were higher than those obtained by either of the two corresponding unmixed infections. As expected for rescue-byrecombination, the fractions of wild type in these progenies were higher than they were in progenies of the same crosses conducted in a permissive host. (The frequencies of the reciprocal (am am) recombinants are not higher in crosses performed in restrictive hosts.) In experiments involving three different late genes. however, the total yields of phage obtained were far below those dealt with in our position-effect survey. Furthermore, crosses between widely separated markers in the (recombinationally) long gene 34 gave greater than 25 % wild type in BB while the total yields of phage obtained were well below those involved in our intergenic tests. We conclude that recombination may reduce, but not seriously so, the sensitivity of our cis-trans tests. 6) Several shortcomings of our analysis were circumvented in the experiments on polarity in the rzz region (see RESULTS). In fact, except for the following two complicating considerations, we would conclude that the A and B cistrons are not co-transcribed. (a) The small cis-trans effect observed cannot be explained away by considerations of finite input (see APPENDIX), since the A protein is probably produced greatly in excess of the amount required for growth of T4 in lambda

13 TRANSCRIPTONS IN PHAGE T4 169 lysogens. (b) We have no evidence for T4 that polarity mutations can be detected in other than the first gene of a transcripton. In several cases where an amber in gene n has a polarity effect on gene nfl and nf2, we tried but failed to demonstrate polarity of ambers in gene nfl upon gene nf2 (e.g., see the tests in Table 1 ). Therefore, the small cis-truns effect we do see in the rll region could be the result of polarity imposed by nonsense mutations in a gene distal to the first one in a multicistronic transcripton. Remarks on the directions of transcription in T4: Our results combined with results of others (see Figure 1 ) reveal that ( 1 ) some genes are transcribed clockwise, others counterclockwise; (2) all the genes known to be transcribed clockwise are in one arc, and the counterclockwise ones are in the other arc of the linkage map; (3) the two arcs may correspond to the major early and late regions of the T4 genome. Points (2) and (3) require comment. Our total knowledge of transcription directions, summarized in Figure 1, includes directions for 24 genes. However, these are not 24 independently established cases, but, in fact, represent only 11 determinations of direction. We can calculate the probability that, by chance alone, the 3 counterclockwise cases will fall in a single run in order to decide whether conclusion (2) should be taken seriously. The 11 determinations define 11 arcs on the circle, each of which marks one end of a set of any three transcriptons. The probability that, by chance alone, such a set will in fact be our set of 3 counterclockwise cases is given by (3/11) x (2/10) X (1/9). Thus the total probability that the three counterclockwise cases will fall in a single run is 6/90. Though this value exceeds the conventional 5 % level for significance, we think conclusion (2) will stand. Of all the genes for which we have knowledge of transcription direction, the 5 which are transcribed counterclockwise are known to be transcribed early. (See BAUTZ et al and KASAI et al for evidence of the early transcription of the e and the rll genes.) Phages amber in genes 44 or 45 fail to make DNA in amber-restrictive hosts, so those genes must also be transcribed early (EPSTEIN et al. 1963). The other 19 genes, for which clockwise transcription has been shown, many be transcribed only late. Our work fails to rule out the simple possibility that in T4 those genes which are transcribed only late are transcribed off one DNA chain, whereas those which are transcribed both early and late are transcribed off the other. This possibility receives support also from the work of GUHA and SZYBALSKI (1968). Our thanks to GEORGE STREISINGER and ENZ~ Russo for help at critical moments, to ILGA LIELAUSIS, BOB EDGAR, SEWALL CHAMPE, JAN DRAKE and SYDNEY BRENNER for providing mutant strains, and to JON KING for communicating unpublished results. The International Laboratory of Genetics and Biophysics in Naples provided hospitality during the preparation of the manuscript, for the typing of which I am pleased to thank Mrs. SUZANNE HAFKIN. SUMMARY We have surveyed the T4 genome for polarity mutations as manifested by intergenic cis-trans position effects. Results of the analysis of the discovered cases provided information on the direction of transcription relative to the linkage map,

14 170 F. w. STAHL et al. bringing to 24 the total number of cases for which transcription direction is known in T4. When coupled with results of others, the data lend support to the idea that those genes of T4 which are transcribed only late are transcribed off one DNA chain while those which are transcribed both early and late are transcribed off the other. LITERATURE CITED ABRAMOWITZ, M. and I. A. STEGUN, (Ed.), 1964 Handbook of Mathematical Functions. Natl. Bureau Standards Applied Math. Ser. 55. U. S. Govt. Printing Office. BAUTZ, E. K. F., T. KASAI, E. REILLY and F. A. BAUTZ, 1966 Gene-specific mrna, 11. Regulation of mrna synthesis in E. coli after infection with bacteriophage T4. Proc. Natl. Acad. Sci. U. S. 55: BENZER, S., 1961 On the topography of the genetic fine structure. Proc. Natl. Acad. Sci. U. S. 47: BENZER, S. and S. P. CHAMPE, 1952 A change from nonsense to sense in the genetic code. Proc. Natl. Acad. Sci. U. S. 443: CRICK, F. H. C.. L. BARNETT, S. BRENNER and R. J. WATTS-TOBIN, 1961 General nature of the genetic code for proteins. Nature 192: EPSTEIN, R. H., A. BOLI.E, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE LA TOUR, R. CHE- VALLEY, R. S. EDGAR, M. SUSSMAN, G. H. DENHARDT and A. LIELAUSIS, 1963 Physiological studies of conditional lethal mutants of bacteriophage T4.D. Cold Spring Harbor Symp. Quant. Biol. 28: GUHA, A. and W. SZYBALSKI, 1968 Fractionation of the complementary strands of coliphage T4 DNA based on the asymmetric distribution of the po1y.u and polyu,g binding sites. Virology 34: JACOB, F. and J. MONOD, 1961 Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3: 31g356. KASAI, T., E. K. F. BAUTZ, A, GUHA and W. SZYBALSKI, 1968 Identification of the transcribing DNA strand for the rzz and endolysin genes of coliphage T4. J. Mol. Biol. 34: NAKATA, A. and F. W. STAHL, 1967 Further evidence for polarity mutations in bacteriophage T4. Genetics 55: SARABHAI, A. S., A. 0. W. STRETTON, S. BRENNER and A. BOLLE, 1964 Co-linearity of the gene with the polypeptide chain. Nature 201 : S~CHAUD, J. and G. STREISINGER: 1962 Phenotypic expression of recombinants arising in phageinfected bacteria. Virology 17: STAHL, F. W. and N. E. MURRAY, 1966 The evolution of gene clusters and genetic circularity in microorganisms. Genetics 53: STAHL, F. W., N. E. MURRAY, A. NAKATA and J. M. CRASEMANN, 1966 Intergenic cis-trans position effects in bacteriophage T4. Genetics 54: STREISINGER, G., J. EMRICH, Y. OKm.4, A. TSUGITA and M. INOUYE, 1968 Direction of translation of the lysozyme gene of bacteriophage T4 relative to the linkage map. J. Mol. Biol. 31: