the - genetics · high negative interference and recombination in bacteriophage t5 * barbara north...

HIGH NEGATIVE INTERFERENCE AND RECOMBINATION IN BACTERIOPHAGE T5 *

BARBARA NORTH BECKt

Department of Genetics, University of Washington, Seattle, Washington 98195

Manuscript received May 5,1980

ABSTRACT

The process of close recombinant formation in bacteriophage T5 crosses has been studied by examining the structure of internal heterozygotes (HETs), the immediate products of recombination events. The T5 system was chosen because it permits the study of internal heterozygotes exclusively, thus avoiding the ambiguities inherent in previous studies with T4. The heterozygotes were obtained by the nonselective screening of progeny phage in a prematurely lysed sample from an eight-factor cross. The molecular structure of each HET was inferred from the strand genotypes displayed among its progeny. This inves- tigation presents unequivocal evidence that both overlap and insertion HETs are intermediates in recombinant formation and that insertion HETs are a significant source of close double recombinants. There is evidence suggesting that mismatch repair of overlap HETs could be the source of close triple exchanges. Thus, a significant part, and perhaps all, of the high negative interference for close-marker recombination observed in this system is a direct consequence of the fine structure of the recombinational intermediates. These findings are compatible with recombination models proposed by others, in which a single branched intermediate can give rise to HETs of both the overlap and insertion types.

RECOMBINATION of closely linked markers is characterized in many sys- tems by an excess of multiple-exchange progeny over the number expected

under the assumption that the multiple exchanges are combinations of independent, single-exchange events. This excess of multiple exchanges has been designated high negative interference (HNI) . These experiments with bacteriophage T5 were undertaken with the idea that examining the process of close- marker exchange could elucidate the physical basis of HNI. Specifically, we want to know how two close exchanges are correlated: Are they the result of one molecular event, or is there a localized stimulation of recombination produced by the occurrence of one exchange that then promotes the occurrence of a second exchange? If the two exchanges do occur in a single molecular event, what is the nature of this event? One can approach these questions in the T5 system by examining the structure of internal heteroduplex heterozygotes (HETs) .

* Submitted by B. N. BECK to the University of Washington in partial fulfillment of the requirements for the Ph.D.

t Resent address: Department of Immunology, Mayo Clinic, Rochester, Minnesota 55901. degree.

Genetics 96; 2541 September, 1980

26 B. N. BECK

The T5 system is advantageous for these studies because, unlike phage A, it produces HETs at a hequency adequate for experimental purposes and because it has a linear map iE contrast to the circularly permuted map of T4. Although the T5 genome is terminally redundant, both genetic (FISCHHOFF, MACNEIL and KLECKNER 1976) and biochemical (RHOADES and RHOADES 1972) evidence indicates that the redundancy is constant. By using markers located in the single- copy region of the genome, one can eliminate the possibility of recovering terminal redundancy heterozygotes and restrict the analysis to loci that will be heterozygous only if included in heteroduplex regions. HETs for these loci should be the immediate products of recombination events. In the present studies, HETs for amber mutations in various genes were obtained by nonselective screening of progeny from an eight-factor cross. From the two strand genomtypes of a HET one can deduce its molecular structure.

Two basic types of internal HET structure have been proposed (LEVINTHAL 1954): (1) an overlap structure, in which markers flanking the heterozygous region are recombined and (2) an insertion structure in which the flanking markers are in the parental configuration (see Figure 1 ) . The hypothesis being tested is that close multiple exchanges (and consequently HNI) are produced

GENOTYPE Structure of DNA Molecule

Parent 1

Parent 2

Overlap HET

- + - +- + - +

+ - + - + - + -

( yields two single recombinants) -+-H+-+-

Overlap HET with mismatch repair (yields one triple recombinant and one single recombinant) - + - H - - + -

Insertion HET (yields one double recombinant and one parental )

- + - H - + - +

- + - + - + - + -

- + - + - -(+I- + -

- + - + - + - + - FIGURE 1 .-Types of heterozygote structures. The parental genotypes and representative

heterozygote genotypes are illustrated. The type of recombinant that each HET will segregate upon replication is indicated. -/- stands for the wild-type allele for the given locus; - stands for the amber allele; H stands for both alleles being present, i.e., the locus is heterozygous. An allele symbol in parentheses means that that allele has been removed by mismatch repair.

HNI AND RECOMBINATION IN T5 27

by the occurrence of more than one genetic exchange in a single molecular recombination event, defined as an event producing a single continuous region of heteroduplex. Clearly, an insertion HET will segregate a double-exchange and a parental genotype upon replication. The segregation products of overlap HETs are two different single-exchange genotypes. However, overlap HETs may contribute multiple-exchange progeny as well, if a mismatch-repair pathway exists. As diagrammed in Figure 1, excision and repair of a mismatch within the heteroduplex region of an overlap HET will result in one triple-exchange strand and one single-exchange strand. Thus, insertion structures can produce close double exchanges, and overlap structures can produce close triple exchanges; the relative frequencies of these exchange classes among HETs will distinguish the con- tributions of the respective pathways.

In the experiments with T5, it will be shown that insertion HETs are a major source of double recombinants for closely linked markers and that overlap HETs accompanied by mismatch repair may also produce triple recombinants. It appears that these two mechanisms can account for the high negative interference observed in this system. These observations will be discussed with reference to a proposed molecular intermediate in recombination.

MATERI.4LS A N D M E T H O D S

Media: Hershey's nutrient broth (H-broth) (CHASE and DOERMANN 1958) was used as the medium for all crosses, assays and for the growth of all bacterial cultures. The top- and bottom- layer agar for plates was the same as that of CHASE and DOERMANN (1958), except that sodium citrate was always omitted and the bottom-layer agar contained only 10.5 g Bacto-agar per liter. All of these media were adjusted to p H 6.8-7.1 and CaC1, was added to 0.001 M just before use.

Bacteria: The Escherichia coli strains used are from the collection of A. H. DOERMANN. They are listed in Table 1 along with their pertinent characteristics. Strains F and HR34 were originally obtained from Y. T. LANNI. All stocks were grown on CR63. Crosses were done in HR34, while all platings of stock assays and of cross lysates were done on a 1:l mixture of HR34 and CR63 exponential cultures. These plating bacteria were prepared by separately subculturing overnight

TABLE 1

Bacterial strains

Strams Characteristics Reference

F Nonpermissive for am mutants. Allows LANNI (1958) fast adsorption of T5.

Derived from F. Permissive for am mutants. Allows fast adsorption of T5.

HR34 LANNI and LANNI (1966)

CR63 Permissive for am mutants. Allows EPSTEIN et al. (1963) only poor adsorption of T5. Suppresses T5 multi-am much better than HR34.

S treptomycin-resistant derivative of F. F/S

HR34/S Streptomycin-resistant derivative Isolated in this lab

Isolated in this lab from F.

of HR34. from HR34.

28 B. N. BECK

aerated cultures at a 1:50 dilution for 2.5 hr a t 30". and then further concentrating the cells 4-fold by centrifugation to 8 x 108 cells/ml. The plating bacteria were kept in an ice bath until used.

Phage stocks: The T5 phage stocks used in these experiments were constructed from single amber mutant stocks that came from 3 different laboratories. The first capital letter in each mutant designation specifies the laboratory in which it was isolated: L (LANNI), M (McCOR- QUODALE), D ( DOERMANN).

In order to eliminate any loosely linked additional mutations from the stocks, the amber mutants 50M-N, 23cM-N and 135aM-N were rescued from UV-irradiated samples (dose = 25 phage lethal hits) of the original stocks. This was done by crossing the irradiated phage to unirradiated T5+ and then testing the progeny phage particles for the original amber alleles. Fresh stocks were made from single plaque isolates of the rescued mutants and designated M-N to distinguish them from the original mutants. The am23D-N mutation was recovered from among the progeny of a cross to T5+ of a multi-amber stock containing 23D. Both am23D-N and am23D have the same amber mutation, but differ in their adsorption properties; more than 95% of the phage in the 23D-N stock adsorb to F at 37" in 10 min, whereas only 50% of the phage in the 23D stock do.

All stocks were grown on CR63 as plate stocks (ADAMS 1959). They were tested for adsorption properties, since stocks frequently acquired mutations conferring adsorption defects. Only stocks in which at least 90% of the phage adsorbed to F or HR34 within 10 min under standard conditions were used in crosses.

Independent maps of the D and L mutants (A. H. DOERMANN, personal communication) and of the M mutants (MCCORQUODALE 1975) had been previously constructed. The maps were combined in the course of this study. All the mutants are in separate complementation groups in the D linkage group of MCCORQUODALE. The m 4 7 D mutation is the marker closest to the end of the D linkage group near the A (the terminally redundant) linkage group; am38D is near the other end of the D linkage group, in the vicinity of MCCORQLJODAI.E'S D4 and D5 genes. The marker arrangements of the multiple-mutant stocks are illustrated in Figure 2. In the earliest experiments, the outside markers am47D and am38D were not included in the stocks.

Cross procedure: The host bacterial culture for a cross was prepared by subculturing an overnight aerated broth culture at a dilution of 1: lOOO in broth at 37" to a titer no greater than 2 x IO7 cells/ml. It was then concentrated to about 2 x 108 cells/ml by centrifugation, and NaCN was added to 0.002 M. The cross was begun 2 to 3 min later by mixing equal volumes of the parental phage mixture and the bacterial culture. The parental phage mixture contained equal numbers of the two parental types at a titer sufficient to give a total multiplicity of infection of IO . Adsorption was allowed to proceed for 10 min at 37O, at which time anti-T5 serum was added to inactivate the unadsorbed phage. At 15 min, the adsorption mixture was diluted 500-fold to the growth tube to release the cyanide inhibition and the incubation was continued at 37". Starting at 30 min (15 min after cyanide release) and every 5 min thereafter until 60 min, a growth tube sample was diluted into broth and CHC1, to stop further growth and to induce lysis ( S ~ H A U D and KELLENBERGER 1956). These constitute the premature-lysis samples. They were incubated for a further 30 min to ensure lysis of all the bacteria and then were assayed for the number of mature phage present per infected bacterium at the time of CHC1, treatment. The growth tube (the normal-lysis sample) was lysed by the addition of CHCI, at 80 min, which is after the normal latent and rise periods of T5 infection have been completed (LANNI 1960). Recombination was analyzed in one of the prematurely lysed samples and in the normal lysis sample.

Heterozygote search and analysis. The 2 strand genotypes of a HET provide information about the molecular recombination event that gave rise to the HET. In order to allow reasonable inferences to be made about the strand genotypes of a HET, and hence, about the nature of the molecular recombination, it is important that the genomes involved have previously undergone very few (if any) exchanges. For this reason, the HET search was conducted on a prematurely lysed sample of a cross. As described above, several samples of the growth tube of a cross were taken and prematurely lysed during the latent period. The sample of progeny phage taken at

HNI A N D R E C O M B I N A T I O N IN T5 29

Parent I

a m t a m i- om + I I I I I I I

am t I

I , 1 1 I

Parent 2

+ om + am -+ om + am I I I I I , I

I I I I I , I I

II 111 I V V V I VI1 I

40 0 5.4 4.8 1.7 11.0 20.0 28 0

47D

FIGURE 2.-The marker arrangement of the multiple-mutant stocks. The intervals are designated by Roman numerals which are used to refer to particular intervals in both the text and tables. The interval sizes are given in centimorgans as calculated by application of the FELSEN- STEIN mapping function (see MATERIALS AND METHODS). The sizes given are those for the normal- lysis time.

40 min after phage addition (25 min after release from the cyanide inhibition) was chosen for the HET search because it was the first sample to contain enough progeny to analyze (6 mature phage per infected bacterium).

To identify heterozygous phage, it was necessary to test several offspring from one phage particle that came from a cross t o determine whether that phage was a heterozygote. If so, it would segregate both the am+ and am alleles for any given locus. The phage suspensions obtained by prematurely lysing samples from the growth tube of a cross were adsorbed for 10 min at 37" to an exponential culture of HR34 at an approximate multiplicity of 10-6 to ensure only single infections. Samples were then plated on a mixed lawn of exponential phase HR34 and CR63 to give 10 to 20 plaques per plate. After 4.5 hr incubation at 37", when most plaques were visible but still quite small, every plaque more than about 4 mm from its neighbors was cut out and resuspended in H-broth plus CHC1,. Each plaque resuspended will contain the progeny, after a few rounds of growth, produced by a single phage. The genotype of this phage will be deduced from the genotypes of its progeny, which will be determined by the replication method (discussed below). For the initial screening, 10 progeny plaques from each resuspension were tested. When more than one genotype appeared among the 10 progeny plaques from a single resuspension, the original infecting phage was tentatively classified as a heterozygote. Sixty more plaques from the same suspension were then tested to confirm the heterozygosity. The 2 strand genotypes of a HET were deduced from the relative frequencies of the various genotypes among the progeny from the HET. Knowing the strand genotypes allows one to infer the most probable molecular recombinational event that could have produced the heterozygote, as illustrated in Figure 1.

Genotype analysis: Phage genotypes were identified by the replication procedure of DOER- MANN and BOEHNER (1970) adapted for use with T5. Plaques were copied onto lawns of CR63. The top layer of test plates (150 mm diameter) contained 0.25 ml of the test phage at IO8 phage/ml, 0.4 ml overnight aerated bacteria and 5 drops streptomycin sulfate at 5 mg/ml. For

30 B. N. BECK

the mutants am47D, am3D and am23aM-N, only F/S was used, while for am50M-N, am23D-N, aml35aM-N, amlL and am38D, a 50:l mixture by volume of F/S and HR34/S was used. These conditions always gave unambiguous tests.

Calculations Interference: For double exchanges, the interference index i has been calculated as the ratio

of the observed frequency of double recombinants to the expected frequency. The expected frequency is the product of the observed frequencies of exchanges in the 2 component intervals. For triple exchanges, the interference index i for the third exchange has been calculated as the ratio of the observed frequency of exchanges in the third interval among phage already doubly recombined to the observed frequency of exchanges for that interval in the unselected population. In this way, the interference index calculated will reflect only the additional interference for the third exchange.

The mapping function: Due to the occurrence of high negative interference in this system, recombination frequencies are not a good measure of map distance between loosely linked genes. In order to obtain better estimates of the distances, recombination frequencies were converted into map distances by a mapping function, derived by FELSENSTEIN (1979), which corrects for the effect of negative interference, The use of this function to generate map distances is justified on the basis that it generated internally consistent distances. Two sets of recombination frequency data were produced in this study for the same set of intervals: the frequencies observed for the premature-lysis sample and those observed for the normal-lysis sample. Both sets of data can be used to estimate the interval map distances by assuming that the ratio of premature- lysis map distance to normal-lyGs map distance is the same constant factor for each interval. There is no a priori reason to assume that the level of interference should be the same for the 2 samples, so that a value of K ( K is the interference index for an arbitrarily small interval) must be estimated separately for each. Then, one can estimate the seven interval map distances (using the mapping function), K for the premature-lysis sample, K for the normal-lysis sample and F , the map ratio factor, by computer iterative techniques. In this process, various values for these 10 parameters are tried in order to find those that best fit the 2 sets of recombination data simultaneously. The fit is judged by a chi-square test, summing the values of chi-square with 4 degrees of freedom (14 independent data points .-7 map distances -2 K values -1 map ratio factor). The map d'stances estimated for the normal-lysis sample are given in Figure 2. The estimates of K for the premature-lysis and normal-lysis samples are 1.6 and 1.35, respectively. The map ratio factor (premature-lysis map distance + normal-lysis map distance) is estimated as 0.54. The chi-square value obtained with this set of parameters was 7.52, which is not significant at the 5 % level. We conclude that the data are not inconsistent with the set of foregoing assumptions and that the use of the mapping function appears justified.

RESULTS

Cross parameters: In the cross used for the HET analysis, the actual multi- plicities of infection were 7.5 and 8.2 phage per bacterium of parents 1 and 2, respectively (Figure 2). The premature-lysis sample taken at 40 min (25 min after release from cyanide inhibition) was used in the HET analysis because it was the first that contained enough progeny phage to analyze. This sample contained an average of six phage per infected bacterium; whereas, the normal-lysis sample (taken at 80 min) contained an average of 850. The heterozygote studies were begun on samples from a cross of two parents identical to the ones diagrammed in Figure 2 except that the markers 47D and 38D were not included. The premature-lysis sample from this cross was taken when there were 15 mature phage per infected bacterium. The 13 HETs identified in this sample are included in the analysis with the 65 from the later cross.

IgNI A N D RECOMBINATION IN T5 31

TABLE 2

Recombination frequencies and map distances

Recombination frequency ~- Map distance$ Interval Premature lysis' Normal lysis+ (in centimorgans)

I 0.131 0.193 28.0 I1 0.156 0.261 40.0 I11 0.026 0.054 5.4 IV 0.027 0.045 4.8 V 0.004 0.022 1.7 VI 0.052 0.101 11.0 VI1 0.096 0.152 20.0

~~~~~~~ ~

* These frequencies are based on the 1105 phage (2210 strands) analyzed in the HET search. + These frequencies are based on the 1008 phage analyzed in the normal-lysis sample. $ The map distances were calculated according to the mapping function described in MATERIALS

AND METHODS. They are given for the time of normal lysis only.

Table 2 lists the recombination frequencies measured for the seven intervals marked in the first cross described above, together with the map distances that were derived from them in the manner described in MATERIALS AND METHODS.

The recombination frequencies are given for both the 40 min premature-lysis sample (those phage analyzed in the search for heterozygotes) and the normal- lysis sample. It can be seen that less recombination has occurred at the time of premature lysis than at the time of normal lysis. This has been shown previously for T2 (LEVINTHAL and VISCONTI 1953) , T4 (DOERMANN 1953), and X (WOLL- MAN and JACOB 1954). It indicates that progeny genomes can mate repeatedly before they are withdrawn from the mating pool and matured (VISCONTI and DELBRUCK 1953). Those withdrawn early have, on average, experienced fewer matings.

High negative interference: The interference index, i, is a useful measure of the statistical dependence or independence of exchange events. Values of i less than one indicate an inverse correlation of exchanges, i.e., one exchange inhibits the occurrence of a second exchange, as in classical chiasma interference in Drosophila. An interference index value of one indicates independence of exchanges, and values greater than one indicate that exchanges are positively correlated, i.e., that the occurrence of one exchange increases the probability of the occurrence of a second exchange. The factor by which the probability is increased is given by the interference index, also referred to as the correlation factor.

The recombination data obtained from the 40 min premature-lysis sample and from the normal-lysis sample of the same cross were examined with regard to the frequency of occurrence of multiple-exchange progeny. In Table 3, the interference index for each pair of intervals has been calculated as described in MATERIALS AND METHODS. It is readily apparent that exchanges in most pairs of intervals are slightly correlated (1 < i < 2), which indicates a small excess of double exchange progeny over the number expected. This slight correlation (sometimes called low negative interference) is a statistical consequence of the

32 B. N. BECK

TABLE 3

The interference index for all pairs of intervals

First exchange Second exchange interval interval __

I

I1

I11

IV

V

VI

I1 I11 IV V VI VI1 I11 IV V VI VI1 IV V VI VI1 V VI VI1 VI VI1 VI1

Size of intervening interval'

InterFereme index at Premature N o r m a l

lysist lysis$

___

0 40.0 45.4 50.2 51.9 62.9 0 5.4

10.2 11.9 22.9

0 4.8 6.5

17.5 0 1.7

12.7 0

11.0 0

1.6 (71) 0.8 (6) 0.8 (6) - (0) 1.2 (28) 1.5 (41) 1.9 (17) 0.6 (6) 1.4 (2) 1.1 (20) 1.2 (40) 1.3 (2) 8.7 (2)

1.4 (8) 4.2 (1) 1.6 (5) 1.4 (8) 4.4 (2) 2.4 (2) 2.6 (29)

3.3 (IO)

1.2 (59) 1.4 (15) 0.9 (8) 1.6 (7) 1.1 (21) 1.1 (33) 1.6 (22) 1.8 (21) 1.4 (8) 1.0 (26)

5.7 (14) 4.2 (5) 1.3 (7)

7.0 ( 7 )

1.7 (12)

1.2 (40)

1.2 (10)

1.1 (5)

4.9 (11) 1.8 (8) 1.4 (22)

* Size is given in centimorgans of map distance at normal lysis, calculated as described in

+ These data are from the 1105 phage (2210 strands) analyzed in the HET search. The num-

$These data are from the 1008 phage analyzed in the normal-lysis sample. The number in

MATERIALS A N D METHODS.

ber in parentheses is the number of double-exchange strands.

parentheses is the number of double-exchange phage.

heterogeneity of mating experience among the progeny genomes, as described by VISCONTI and DELBRUCK for T2 (1 953). More significantly, however. it can be seen that for neighboring, short intervals (intervals 111, IV and V), the calculated interference index is much higher (3 < i < 9), indicating much stronger correlations of exchanges than those due to mating experience. Nor can these higher values of i, indicating a substantial excess of multiple-exchange progeny over that expected, be ascribed to random fluctuation in the data. A chi-square test indicates significant ( p < 0.005) deviation from a random distribution of exchanges. This statistical test of randomness was performed only on the normal- lysis data; the small size of the double-exchange classes in the premature-lysis data precludes application of this test.

This high correlation of exchanges in neighboring, short intervals is the phenomenon first described by STREISINGER and FRANKLIN (1952) in phage T2 and later extensively characterized by CHASE and DOERMANN (1958) in T4 and by AMATI and MESELSON (1965) in A. CHASE and DOERMANN referred to it as high negative interference (HNI), whereas AMATI and MESELSON preferred the

HNI -4ND RECOMBINATION IN T5 33

term localized negative interference. The characteristic of high (or localized) negative interference that clearly distinguishes it from low negative interference, in addition to its magnitude, is its dependence on distance. Both CHASE and DOER- MANN and AMATI and MESELSON observed that the shorter the distance between the markers studied, the higher the correlation (the greater the excess of multiple exchanges). It is this distance-dependence of the phenomenon which leads t o the hypothesis that HNI may be a consequence of the physical process of re- combinan t f o m a t ion.

HNI, with its distance-dependent property, can be simulated by the improper inclusion of heterozygotes in the wild-type recombinant classes. Under standard genotype testing procedures, heterozygotes have the opportunity to segregate progeny homozygous for the wild-type allele during growth of the plaque before testing. Due to the dominance of the wild-type phenotype over the amber phenotype, tests will reveal the presence of the wild-type allele only. Mature progeny genomes having heterozygous regions may then appear to have experienced more exchanges than they actually have. Thus, while the probability of heterozygosity at any locus is roughly the same, HETs will make a disproportionately large contribution to the total number of exchanges in short intervals since exchanges are rare in these intervals. However, in these experiments with T5, all of the HETs in the premature-lysis sample were identified and assigned to the proper recombinant classes. Thus, the HNI observed in the premature-lysis sample is free of any artifact due to misclassified HETs. When recombination frequencies were recalculated for the normal-lysis sample, scoring only the double-mutant classes of recombinants rather than scoring both wild-type and double-mutant, HNI still persisted (data not shown). This source of artifactual HNI was ruled out in the same manner for T4 by DOERMANN and PARMA (1967).

From the recombination data of this cross, it is possible to determine whether there is HNI also for triple exchanges. As mentioned in MATERIALS AND METHODS,

the interference index, i, for the third exchange has been calculated as the fol- lowing ratio: the observed frequency of exchanges in the third interval among phage already doubly recombined divided by the observed frequency of exchanges in that interval in the unselected population. This value expresses the increased probability (over that of a progeny genome selected at random) that a genome, which has already experienced two exchanges, will experience yet a third. It thus reflects only the additional interference for the third exchange. It must be noted that the actual value of this number will depend on which interval, of a set of three, is chosen as the third interval.

The interference index for the third exchange has been calculated from the normal-lysis time recombination data. The values of i calculated for the five possible sets of three adjacent intervals are displayed in Table 4. The interference index calculated for nonadjacent sets of three intervals was generally less than three (data not shown). It is clear that, as for HNI for a second exchange, HNI for the third exchange reaches substantial levels when small intervals (111, IV and V) are adjacent. It can also be seen that, in general, the highest interference is observed when the center interval is chosen as the third exchange interval.

34 B. N. BECK

TABLE 4

T h interference index for the third exchange at normal lysis'

Set of three adjacent exchange intervals

Interference index when third exchange interval is. No. of triple-exchange

Left Center Right progeny

I, 11, I11 1.9 2.0 2.6 8 11,111, I V 2.2 10.6 8.1 8 111, IV, v 7.9 13.3 9.7 3 IV, v, VI v, VI, VI1

6.1 27.3 4.2 3 8.3 6.6 2.4 4

* The interference index for the third exchange is calculated as described in MATERIALS AND METHODS. The number calculated is dependent on the interval chosen as the third-exchange interval.

This is understandable since the initial selection fo r two exchanges, one on each side of this interval, increases the likelihood that this whole segment of the genome has been available for recombination. Triple-exchange progeny were quite rare in the premature-lysis sample, so that calculation of interference indices from those data would provide little insight. However, from the normal- lysis data, it is clear that HNI is observed for a third exchange, in addition to that characteristic of a second exchange.

HET frequency: The observed frequency of heterozygosity per locus per phage in the premature-lysis sample was 0.009. The individual locus frequencies ranged from 0.004 for 3D to 0.015 for 38D, but a chi-square test showed the distribution of HETs was not different from random at the 5% level of signifi- cance. Seventy-eight out of 1,465 phage tested had one heterozygous region. One phage was heterozygous in two regions, 47D at one end and 1L and 38D at the other end of the marked region. This HET has been excluded from the analysis because unambiguous assignment of strand genotypes was not possible. Fifteen of the 78 HETs were heterozygous for two o r three markers. It is assumed that these multi-marker HETs are single stretches of heteroduplex DNA; this seems justified since all the multi-marker HETs involved adjacent markers except for the one mentioned above. Since the observed frequency of heterozygous regions per phage is about 0.05, one would expect about tw:, phage with two HET regions of independent origin in a sample of this size; the observations are not significantly different from expectation.

HET structure: In theory one can propose both an overlap structure and an insertion structure for every HET. An overlap structure can be converted to an insertion structure (and uim uersa) by assuming that one of the participating strands has experienced an exchange previously. If one assumes also that repair of heterozygous (mismatched) base pairs can occur, then the number of structures imaginable for any given HET increases. Thus, in order to make an assignment of a particular structure to a given HET, consideration has to be given to the relative probabilities of various events.


Premature cross lysates were used as the source of phage for the HET search, since, on average, these phage have experienced fewer exchanges than have phage recovered in the normal cross lysate. This choice of HET source allows one to assume that most of the strands that will have participated in the formation of HETs will have been of one or the other parental genotype. One can estimate the likelihood of participation by a recombinant strand from the relative frequency of that genotype.

In some cases, which will be considered in more detail below, forming a particular HET structure with strands of the parental genotypes would require mismatch repair of part of a heterozygous region. It is not possible to estimate the likelihood of occurrence of such mismatch repair, since there is no direct evidence that such a repair pathway exists in T5-infected cells. However, one can assess the probability of formation of a HET region of a particular length (which would then be partially removed by the repair mechanism) and compare this with the probability of formation of the alternative HET structure requiring participation of a recombinant strand. It is important to keep in mind that this comparison cannot be strictly quantitative, for two reasons: First, from these genetic data, one cannot draw exact conclusions about the length of heterozygous regions formed during the recombination process. The heterozygous regions observed in mature phage may have been shortened by a mismatch-repair process. Second, without independent information about the existence and frequency of occurrence of mismatch repair, it is not possible to evaluate quanti- tatively the potential contribution from this pathway. However, even though unequivocal assignment of HET structures cannot always be achieved, the analysis of HET structures can reveal general trends in HET types. These trends provide some insight into the recombination process.

Of the total of 78 HETs, 46 did not include either 47D or 38D (the outermost markers) as heterozygous loci; thus, both ends of their heterozygous regions are identifiable. For each of these 46 HETs, one can assign a most probable structure, based on the considerations discussed above. The major classes of HET structure are diagrammed in Figure 1 and the assignment of HETs to these classes is set out in Table 5. Of the 46 HETs, 26 can be unambiguously assigned t o the overlap class and 11 to the insertion class. Six of these 37 have an additional crossover at a considerable distance from the heterozygous site, which presumably occurred during an independent recombination event. The remaining nine gf the 46 HETs, diagrammed in Figure 3, cannot be unambiguously classified, although in some cases there are good reasons to prefer one structure over the other.

HETs 6-21, 10-117 and 11-24 are very likely overlap structures formed by one parental strand and one recombinant strand. For each of these, an insertion structure formed by two parental strands would require a HET region much longer than any that has been observed (at least 40 CM for 10-117, and at least 45.4 cM for 6-21 and 11-24), the greater part of which would then have to be removed by mismatch repair. I t seems unnecessarily complicated to propose

36 B. N. BECK

TABLE 5

Classes of HETs obserued ~~ ~ ~ ~~~

One-ended (only one end inside the marked region)

Two-ended (both ends inside the marked region) Participating strands Structure of HET

Overlap Two parental strands One parental and one recombinant

Insertion Two parental strands One parental and one recombinant

Indeterminate (see Figure 3)

Total

______.__-

Grand total

32

22 4 9 2 9

46

78

mismatch repair for these HETs. The recombinant strand required for the overlap structure appears with a frequency of 0.065 among the progeny.

For HETs 6-113, 7-82 and 10-208, there are no compelling reasons for pre- ferring one structure over the other. Each of these HETs could have arisen from the interaction of two parental strands and subsequent mismatch repair of part of the heteroduplex region. HETs 6-1 13 and 7-82 would then be overlap struc-

HET number

6-2 1

10-11 7

11-24

6-11 3

7- 02

10-208

12-52

4-107 *

8-104

Genotype

- - H H - + - +

+ + H - + - + -

+ + - t i + - + -

- + t i + + - + -

- + + t i + - + -

- + + t i - + - +

- + + - H - + -

- + H - + +

+ - - + - H - +

Overlap structure

+ - - + - + - + - -

+ + - + - + - + - + + - + - + - + - - + - +

+(-I + - + -

+ - + - - ‘‘7’ 2

- + + - + - + - + - +(-l(+)- + - + - + - - + - + - + + t - (+I (-1 (+) -

I n s e r t i o n structure

-(+I- + - + - +

+( - I+ - + - + -

+( - ) (+ I - + - + -

- + -

+ -

+ - + - + - + + - + - + - + + - + - + - + - + ( - I + - + - + + - - + + - + - + - -

+ - + - + - (+ I ( - )+

+ - - + - + - + - + - + - + -

Preference

0

0

0

?

?

?

I

I

I FIGURE 3.-HETs for which the structural assignment is uncertain. The structure preferred,

based on probability considerations, is indicated as 0 for overlap, I for insertion and ? for either. The allele symbols are the same as in Figure 2.

* This HET came from the first cmss lysate used in the HET analysis, in which the outer markers on each end, um47D and am38D, were not included; thus, the lefimost marked locus in this HET is um3D.


tures and HET 10-208 an insertion structure. The proposed heteroduplex region is the same for all, extending from 50M-N to 23D-N (at least 5.4 cM). HET regions of this size have been observed frequently. The alternative structure for each of these HETs is that formed by a recombinant strand and a parental strand. The required recombinant strand for HET 6-113 occurs with a frequency of 0.014, and that for HETs 7-82 and 10-208 (it is the same one) with a frequency of 0.078. Thus, one cannot come to any definite conclusion about the structures of these HETs.

The last three HETs, 12-52, 4-107 and 8-104, are probably insertions. For HETs 12-52 and 8-104, overlap structures between two parental strands would require heteroduplex formation over regions longer than have been observed, which would then be partially removed by mismatch repair. The length of heteroduplex required is not enormous (at least 10.2 cM for 12-52, and at least 12.0 cM for 8-104); it simply has not been observed. The recombinant strands required for the insertion structures each appear with a frequency of 0.078. For HET 4-107, an insertion structure formed by two parental strands, followed by mismatch repair, appears reasonable, since HET regions of such length have been observed. The recombinant strand necessary for the alternative overlap structure appears with a frequency of 0.026.

From this analysis of HET structures, one can conclude that both insertion and overlap HETs occur in T5, but not with equal frequency ( p < 0.025). Of the 46 HETs to which structures can potentially be assigned, 26 are clearly overlaps and 11 are clearly insertions. Among the remaining nine, three are most likely overlaps and three more insertions, although absolute assignments cannot be made. Thus, it appears that overlap structures occur roughly twice as frequently as insertion structures. Direct evidence for the occurrence of mismatch repair is lacking. No HETs were observed in which there were homozygous loci within otherwise heterozygous regions. Mismatch repair could be expected to produce such structures.

DISCUSSION

The fact that high negative interference is observed in crosses involving tightly linked markers means that such close exchanges, unlike exchanges farther apart, are not statistically independent of one another. The object of this study has been to ascertain the basis for this lack of independence. One can imagine that the exchanges are correlated because they occur concomitantly in a single recombination event, or because the occurrence of one exchange promotes the occurrence of further exchanges in the same region. Two approaches have been used by others to distinguish these possibilities: EDGAR and STEINBERG (1958) asked how many mating events between genomes were required by assessing the effect of alterations in the parent input ratio on double-recombinant production. Their data supported the hypothesis that HNI for double exchanges was primarily due to correlated exchanges in a single mating (recombination) event. KELLEN- BERGER, ZICHICHI and EPSTEIN (1962) analyzed HET structures in the phage A

38 B. N. BECK

system. They found that double recombinants arose primarily from insertion HETs and thus were produced in single interactions between genomes.

The data on T5 recombination presented here also support the hypothesis that multiple exchanges occur in a single molecular recombination event. Table 6 lists the total number of multiple-exchange strands (in which the exchanges were in adjacent intervals) that were observed in the premature-lysis sample and how many of these were observed in HETs. It is immediately evident that all five of the multiple exchanges for the shorter intervals (I11 and IV, IV and V, V and VI) were found in heterozygous phage; none was found in homozygous phage. This must mean that these HET structures are representative of the major ways in which close multiple-exchange strands are produced. This paper presents ample evidence that insertion HETs are formed and, thus, that the insertion HET pathway contributes close double-exchange strands. However, the observation of HNI for triple exchanges, as well as for double exchanges, indicates that there should be a pathway in addition to the insertion HET pathway by which multiple exchanges can occur during one molecular recombination event. NIismatch repair in the heteroduplex region of an overlap HET, which produces one triple-recombinant strand and one single-recombinant strand, would be such a pathway. Two HETs were observed in the premature-lysis sample (6-113 and 7-82, Figure 3) whose structures are compatible with this pathway. However, in the absence of direct evidence that mismatch repair occurs in this system, one can state only that the data are compatible with the existence of such a pathway giving rise to triple recombinants and thereby contributing to HNI.

BROKER and LEHMAN (1971) proposed a molecular pathway for recombination in the T4 system based on an EM analysis of recombining DNA molecules. They specifically proposed the existence of a branched intermediate that could be resolved to either an insertion structure or an overlap structure; thus. one pathway would be responsible for the production of both single and double re- combbants. However, there was no direct genetic evidence for the existence of insertion HET structures. Earlier, Fox (1966) had proposed essentially the

TABLE 6

Multiple-exchange strands at premature lysis

Intervals

I and I1 I1 and I11 I11 and IV IV and V V and VI VI and VI1

Size'

68.0 45.4 10.2 6.5

12.7 31.0

~ ~~

Total number of strands with exchanges

in both intervals Number in HETs

71 4 17 5 2 2 1 1 2 2

29 2

* The size given is the sum of the two interval map lengths (in centimorgans) at the time of normal lysis.


same intermediate in a discussion of the mechanism of genetic transformation in bacteria. At that time, he suggested that his proposed pathway, involving a heterozygous intermediate, might explain bacteriophage recombination as well.

The analysis of HET structures in T4 (and T2) is greatly complicated by the occurrence of terminal redundancy (TR) HETs. Not knowing that TR HETs existed, LEVINTHAL (1 954), studying T2, concluded that it was unnecessary to postulate two HET types; the data were compatible with the existence of only overlap HETs. BERGER (1965) and KVELLAND (1969) performed T4 crosses in the presence of FUdR, which selectively increases the frequency of heteroduplex HETs ( S~CHAUD et al. 1965), and obtained data consistent with the existence of both overlap and insertion HETs. However, the evidence was indirect since specific ctructures could not be assigned to specific HETs. In other experiments with T4, STEINBERG and EDGAR (1962) attempted to ascertain the physical basis of HNI by asking which exchange class predominated in crosses of three closely linked markers flanked by loosely linked markers. They assumed that all recombinants would arise as segregants from HETs that were recombinant for outside markers, i.e., from overlap HETs. They then reasoned that all double exchar,ges among their closely linked markers would be accompanied by a third exchange between the flanking loosely linked markers. This hypothesis predicts that in this system triple exchanges would be more frequent than double exchanges. In fact, the opposite result was obtained: double exchanges were more frequent than triple exchanges. Therefore, some aspect of their hypothesis was incorrect; it is most likely that the assumption of only overlap HETs was in error, in view of the data of BERGER (1965) and KVELLAND (1969) discussed above.

TRAUTNER (1958) investigated HETs in the T1 system, in which TR HETs do not confound the analysis. He proposed that an exchange in an adjacent interval sometimes may accompany the formation of an overlap HET (the end result is equivalent to an insertion HET) in order to explain all the HETs as products of overlap structures. From an analysis of HETs produced in replication-blocked crosses of phage A, WHITE and Pox (1974) proposed that close multiple exchanges most likely are the result of mismatch-repair of overlap HETs. They did not observe recombinants arising from insertion HETs, but this pathway probably was blocked as a result of the replication block (STAHL et al., 1973).

The data presented here on T5 HET structures clearly demonstrate that both overlap and insertion structures are formed in this system, although not with equal frequency; overlaps appeared about twice as frequently as insertions. It should be borne in mind that, since the HETs observed are those that have been matured into progeny phage genomes, they may not be entirely representative of the pool of genomes engaging in recombination. The observed greater frequency of overlap structures may reflect am influence of the maturation process, rather than a bias in the formation of the two types of structure. Nonetheless, it is clear that both types of structures contribute recombinants and that these data are compatible with the recombination intermediate proposed by BROKER

40 B. N. BECK

and LEHMAN. The data of STEINBERG and EDGAR (1962) with T4 and those of TRAUTNER (1958) with T1 are also compatible with this model; it is unclear at the present time whether A recombination follows a similar mechanism. Thus, the T phages, at least, may share a common recombination pathway, in which both insertion and overlap structures are produced from a single intermediate structure.

I thank A. H. DOERMANN for his assistance and encouragement, and J. FELSENSTEIN for the mapping function and numerous consultations on the statistical analysis. This work was supported by Public Health Service grants GM-00182 to the Department of Genetics, University of Washington, and GM-13280 to A. H. DOERMANN.

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Bacteriophages. Interscience Publishers, New York. Localized negative interference in bacteriophage A. Genetics

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recombination. J. Mol. Biol. 81: 1-16. BROKER. T. R. and I. R. LEHMAN, 1971

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High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43 : 332-353.

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The identification of complex genotypes in bacteriophage T4. I. Methods. Genetics 66: 417-428.

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DOERMANN, A. H. and D. H. PARMA, 1967 Recombination in bacteriophage T4. J. Cell. Physiol. 70: (Suppl. 1): 147-164.

EDGAR, R. S. and C. M. STEINBERG, 1958 On the origin of high negative interference over short segments of the genetic structure of bacteriophage T4. Virology 6: 115-128.

EPSTEIN, R. H., A. BOLLE, C. M. STEINBERG, E. KELLENBERGER, E. BOY DE LA TOUR, R. SHEVALLEY, R. S. EDGAR, M. SUSSMAN, G. H. DENDARDT and A. LIELAUSIS, 1963 Physiologi- cal studies of conditional lethal mutants of bacteriophage T4D. Cold Spring Harbor Symp. Quant. Biol. 28: 375-392.

A mathematically tractable family of genetic mapping functions with different amounts of interference. Genetics 91 : 769-775.

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teriophage T4D. Genet. Res. 14: 13-31.

FELSENSTEIN, J., 1979

FOX, M. S., 1966

KELLENBERGER, G., M. L. ZICHICHI and H T. EPSTEIN, 1962 Heterozygosis and recombination

KVELLAND, I., 1969 The effect of homozygous deletions upon heterozygote formation in bac-

HNI AND RECOMBINATION IN T5 41 LANNI, Y. T., 1958

LANNI, F. and Y. T. LANNI, 1966 J. Bacteriol. 92: 521-523.

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RHOADES, M. and E. A. RHOADES, 1972 Terminal repetition in the DNA of bacteriophage T5. J. Mol. Biol. 69: 187-200.

S~CHAUD, J. and E. KELLENBERGER, 1956 Lyse prkcoce, provoquke par le chloroforme, chez les bactkries infectkes par du bactkriophage. Ann. Inst. Pasteur 90: 102-106.

SQCHAUD, J , G. STREISINOER, J . EMRICH, J. NEWTON, H. LANFORD, H. REINHOLD and M. M. STAHL, 1965 Chromosome structure in phage T4. 11. Terminal redundancy and heterozygosis. Proc. Natl. Acad. Sci. U.S. 54: 1333-1339.

STAHL, F., S. CHUNG, J. CRASEMANN, D. FUALDS, J. HAEMER, S. LAM, R. MALONE, K. MCMILAN, Y. NOZU, J. SIECEL, J. STRATHERN and M. STAEL, 1973 Recombination, replication, and maturation in phage lambda. pp. 487-503. In: Virus Research. Edited by C. Fox and S. ROBINSON. 2nd ICN-UCLA Symp. Mol. Biol. Academic Press, New York.

STEINBERG, C. M. and R. S. EDGAR, 1962 A critical test of a current theory of genetic recombination in bacteriophage. Genetics 47: 187-208.

STREISINGER, G. and N. C. FRANKLIN, 1956 Mutation and recombination at the host range genetic region of phage T2. Cold Spring Harbor Symp. Quant. Biol. 21: 103-111.

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WHITE, R. L. and M. S. Fox, 1974 On the molecular basis of high negative interference. Proc. Natl. Acad. Sci. U.S. 71: 1544-1548.

WOLLMAN, E. L. and F. JACOB, 1954 Etude gknktique d'un bactkriophage temper6 #Escherichia coti. 11. Mkchanisme de la recombinaison gknktique. Ann. Inst. Pasteur 87: 674-690.

Corresponding editor: G. MOSIG

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