DISTRIBUTION AND FREQUENCY OF THE rZZ REGION … · 2003. 7. 26. · DISTRIBUTION AND FREQUENCY OF TANDEM DUPLICATIONS OF THE rZZ REGION OF BACTERIOPHAGE T4D DAVID H. PARMA, G. THOMAS

DISTRIBUTION AND FREQUENCY OF TANDEM DUPLICATIONS OF THE rZZ REGION OF BACTERIOPHAGE T4D

DAVID H. PARMA, G. THOMAS HEATH, CHIA-CHUNG CHE

AND J. L. ANNEST

Department of Biology, University of Utah, Salt Lake City, Utah 84112

Manuscript received December 29, 1975 Revised copy received June 27,1977

ABSTRACT

Genetic analyses of 49 duplications of the rll region of bacteriophage T4D suggests that there is a non-random relationship between the end points of duplicated segments, that relaxed packaging restrictions have little if any effect on the distribution of duplications, that segregation is 3-4 times more frequent than normal recombination for the same interval, and that nontandem duplicatims are rare. Extrapolation of the r1231 X rJ101 cross data suggests that the minimum frequency of duplications/genome is 1.7 X 1P6, but possibly 3.4 x IO'.

T A N D E M duplications of the rll region of bacteriophage T4D can be isolated from among the progeny of r1589 x rJi'Ol crosses (WEIL, TERZAGHI and

CRASEMANN 1965; PARMA and INGRAHAM 1970; SYMONDS et al. 1972). These duplications produce approximately equal numbers of triplication and unduplicated chromosomes by asymmetrical pairing and crossing over ( PARMA, INGRA- HAM and SNYDER 1972), Their stability is inversely related to their size, and due to restrictions of T4's headful packaging mechanism, duplication genomes frequently contain a deletion of physiologically nonessential information to compensate f o r their otherwise excessive size (WEIL and TERZAGHI 1970; PARMA, INGRAHAM and SNYDER 1972; HOMYK and WEIL 1974).

The present experiments constitute genetic analyses of 49 duplications and wem undertaken to (1 ) determine the relationship between the left and the right ends of duplicated segments; (2) assess the effect of relaxed packaging restrictions on the distribution of ends; ( 3 ) evaluate in a more quantitative fashion the relationship between stability and genetic length; (4) determine if nontandem duplications occur with a detectable frequency; ( 5 ) estimate the frequency of occurrence of duplications in T4; and (6) provide a collection of genetically defined duplications for use as probes in studying other problems. The results suggest a nonrandom relationship between ends; within limits, the absence of qualitative effects on the endpoint distribution by relaxed packaging restrictions; and a minimum estimate of the frequency of duplications/T4 genome of, perhaps, as high as 5 x Genetics 87: 593-619 December, 1977.

594 D. H. PARMA et al.

MATERIALS A N D METHODS

Strains: Except as noted, procedures and phage and bacterial strains are identical to those of PARMA, INGRAHAM and SNYDER (1972) and PARMA and SNYDER (1973). Phage mutations (Figure 1) and the relevant phenotypes are listed in Table 1. The bacterial strains used in these experi-

TABLE 1

Phenotype and gene of mutants*

Gene Mutation Phenotype

52 ac ac-rll

D region-rllB

rIIB

rIIB-rIlA

r I I A

60

39

amE663 ac41 rNB2226

rdP8

rNB3162

rNB3157 rPB296 r638 r73 rB94 r J l 01 r1589 r1231

rJ87 rHB84 r70 rHB118 r219 rAP129 amE429 amHL626 amE3OO amE1217 amE594 amE416 amE26 amE52 amE566 amE556 amEl204 amNG604 amE205

rl r48 rapid lysis tRNA A33 deletion including the t-RNA region of T4;

approximately 4000 base pairs in length

amber acriflavin-resistant acriflavin-resistant B-terminal deletion; deletes

entire r l l region acriflavin-resistant B-terminal deletion; deletes

all of B and most of A cistron acriflavin-resistant B-terminal deletion; deletes

all of B and the A5d2-A6d segment of A cistron T4B B-terminal B-cistron deletion; overlaps r1589 T4B B-terminal B-cistron deletion; overlaps r1589 T4B B-terminal B-cistron deletion; overlaps r1589 small deletion, does not overlap r1589 amber; rB94 site is deleted by rJlOl small deletion; overlaps r1589 A-B deletion with B function A-B deletion with B function when reading frame

corrected by an appropriate A cistron frameshift mutation

small deletion, covered by r1589 amber; rHB84 is deleted by r1589 point mutation amber point mutation point mutation amber

amber

* All mutants originally isolated in T4B have been cross-reactivated into T4D as described by PARMA, INGRAHAM and SNYDER (1972).

T4 r l l DUPLICATIONS 595

ments are E. coli CR63/hh, CAJ-70, S/6, CR63(Xh), Kl2(h)/S, B and 011'. The abbreviations for these strains are CR/hh, CAJ, S/6, CR(Xh), K/S, B and O l l ' , respectively [see Table 2 of PARMA and SNYDER (1973) for relevant properties]. Phage and bacterial strains were obtained from S. CHAMPE, A. H. DOERMANN, L. GOLD and W. B. WOOD.

Mixed indicator assays: A modification of the R:K plating technique of WEIL, TERZAGHI and CRASEMANN (1965) was used to identify r segregants in single-cycle growth experiments. E. coli B host cells were prepared according to CHASE and DOERMANN (1958), adjusted to 4 x 108 cells/ml in 2 X lor3 M CN-, and mixed with an equal volume of a T4 single-cycle growth lysate adjusted to a titer of 2 x IO4Jml. After a ten-minute adsorption period at 20", the adsorption mixture was diluted IO-fold into T4 antiserum for ten minutes, and 0.1-ml aliquots were then plated on a 1:5 or 1 : l O mixture (by volume) of CR/Ah:CR (Ah) plating cultures.

Nomenclatures: Duplication strains are indicated by the symbol Dp and a three-digit isolation number. The left-to-right map order used here c o r r e s p d s to the clockwise order of the T4 genetic map. The notation r1589. rJ1OZ indicates a duplication in which r15S9 maps in the left half and rJ1M in the right. The converse orientation is noted as r.7201 * ri589. The inclusion of either in parentheses indicates that the order is unknown and/or irrelevant. Functional duplication means a plaque-forming duplication that is able to grow on a lambda lysogen. Segregation frequency is the ratio of r segregants to total progeny in single-cycle growth lysates. The corrected frequency is adjusted for triplication progeny, which are assumed to be inviable and as frequent as r segregants. An insertion duplication is one in which the repeated segment is integrated into the genome at a nontandem position.

RESULTS

Operational classification: The duplications analyzed in the following experiments were isolated from two parallel crosses: those in the 600 series from rJlOl rHB84 r48 A33 X r1589 r48 A33, and those in the 700 series from rJlOl rHB84 r48 x r1589 r48. The crosses differ only in that the former is homozygous for the 4000-base-pair deletion n33, which is located in the tRNA region of the

36 52 D rIIB rIIA 60 39 c1 -

E663 r73 rH664 rHB118 E429 E416 E 566 E556 E205 8262 ac41 rJlOl r70 rAP129 E300 E 26 NG604 El204

I

k1.2-1 *3.9----1 l-3.1+3.3+2.0+2.0-t4.2~+2.1~+4.2+l.341.02+3.0i

t - l 3 . 2 . f - 6 . 2 4 F 2 . 9 1 C 3 . 5 + 5.6 +3.E2+ r1589 l-----8.62'-4.12---1 C3.5-6.5+ rdP6

I-3.8 ' 1

rNB3162 - r636 -

r PB 296 - rN 83157

r1231 - 6.5

FIGURE 1 .-Genetic map of the region from gene 3s to gene 39. Intervals are not drawn pro- portional to genetic size. H U 2 6 and E429 map at or near the same site. Recombination percent is twice the percent wild-type recombinants determined by selective plating. Superscripts indicate the number of times a particular cross was performed. The order of mutants has been determined by three-factor crosses (D. H. PARMA and G. T. HEATH, unpublished data). Sites not ordered with certainty are enclosed in parentheses.


T 4 genome (WILSON, KIM and ABELSON 1972). With the exception of Dp617-1 and Dpdl7-2, each duplication was isolated from a different infective centra. Duplications obtained from different selection plaques are assumed to be of independent origin. Of the 49 duplications, 43 behave as normal tandem duplications, while six are anomalous. To simplify the presentation of results, data for the normal duplications will be presented first.

Based on the position of the left end, PARMA and SNYDER (1973) defined three classes of duplications: Class I, those in which the end is to the rZZ-distal side of the ac locus; Class 11, those in which the end is between the rll distal side of the ac locus and the B cistron; and Class 111, those in which the end occurs within the B cistron (see Figure 3, PARMA and SNYDER 1973). The operational criteria for this classification an2 a duplication's acriflavin-resistance mutation index and its ability to form acriflavin-resistant (ac') triplications when crossed to the acr-rlZ deletion rdP8 (see Figure 4; PARMA and SNYDER 1973). Class I duplications have a lower than normal acriflavin-resistance mutation index and are unable to form mr triplications. Class I1 duplications exhibit a normal mutation index and do form acr triplications, while Class I11 duplications have a normal mutation index but do not form acr triplications.

The ratio of each duplication's ecr mutation index on CR(Xh) to that on CR/Ah is plotted in Figure 2. This ratio compares the mutation index of a duplication to that of all plaque-forming phage (duplications plus segregants) in the same

E l " 3 ;) ,~ z

.01 .03 .05 .07 .09 20 .40 .60 .80 1.0 3.0 ocr MUTATION INDEX on CR(1h) oCr MUTATION INDEX on CR&

FIGURE 2.-Ratios of acr mutation indices. The acr mutation indices of duplication stocks which contained about 50% segregants were measured on CR/U and on CR(Xh). The index on the former is that of all plaque-forming phage (duplications and r segregants), while the index on the latter is for duplications exclusively. The data indicate that, in general, duplications have a slightly lower index than their segregants. The average ratio for seven determinations of a nonduplication control, r48, was 1.01 with a range of 0.81-1.22. The results for a particular duplication are indicated by its isolation number. Note that the scale on the abscissa is nonlinear.

T4 rZZ DUPLICATIONS 597

stock. A ratio of slightly less than one may be expecteic fo r Class I1 and I11 duplications since a duplication’s effective burst size on CR(xh) is smaller than that of the duplication or its segregants on CR/hh. Under stress conditions, such as encountered on acriflavin-supplemented plates, this may result in a somewhat lower efficiency of plating. Forty ratios fall in the range 0.29-1.52 and have a median of 0.60. These duplications are classified as 11’s and 111’s. Three duplications have ratios < 0.05 and are assigned to Class I.

The proportion of progeny able to grow on CR(hh) on acriflavin-supplemented plates from crosses of an ac+ duplication to the ucr-rZZ deletion rdP8 is summarized in Figure 3. Duplications which cannot form acr triplications can produce progeny able to grow on CR (Ah) plus acriflavin by mutating to acr. These progeny should occur with a frequency equivalent to the acr mutation index, or about As expected, a mode occurs in Figure 3 at this value. The duplications repr3sented in this mode are members of Class 111. A second mode appears at 3 x and indicates the formation of acr triplications. The corresponding duplications fall in Class 11.

It should be noted that a Class I1 duplication could be spuriously assigned to Class 111 if the right end is in the portion of the r1589 A cistron that is deleted by rdP8. Dp622 is a case in point. When crossed to rdP8, ucr progeny on CR(hh) appear with a frequency of 2.5 X which is not different from its mutation index. However, the frequency of r.7101 segregants appeared to be unreasonably high for a Class I11 duplication (see Table 2). Consequently, it was examined further by crossing to rNB3162, which, like rdP8, is an ncr-rZZ deletion lacking both A and B function but not extending into the A cistron as far as does r1589. From Figure 3 it can be seen that in crosses to rNB3162, Dp622 produces acr

8 10- 0 z W

3 0 0 0

L L 0

a

5 5. m z 3 z -

n lb-6 ~ x I O - ~ lb-5 5xi0-5 5:10-4

acr PROGENY onCR(Xh) /TOTAL PROGENY on CR(Xh)

r’l 10-3 ~ x I O - ~

i n

~ x I O - ~ lb-5 5xi0-5 acr PROGENY onCR(Xh) /TOTAL PROGENY on CR(Xh)

FIGURE 3.-Frequency of acriflavin-resistant progeny in crosses of acriflavin-sensitive duplications to rdP8. The data for Dp622 are from a cross to rNB3162. The frequency of progeny on C R ( h h ) on acriflavin-supplemented plates in those crosses in which it is about l e 5 or less does not differ from the parental duplication’s mutation index to acriflavin resistance. Production of ac7 progeny on C R ( h h ) with a frequency of l e 3 o r higher is due to the formation of acr triplications. The results for a particular duplication are indicated by its isolation number.

598

triplications with a frequency of 3 x That the right-hand end of Dp622 is in fact in the A cistron has been confirmed by additional mapping experiments (see Table 3 and Figure 6).

In summary, the 600 series contains two Class I, thirteen Class 11, and seven Class I11 duplications, while the 700 series has one, ten and ten, respectively.

Orientation of r1589 and rJlOl: The order of r1589 and r.7201 in each duplication has been determined by crossing an acf (r2589.r.7101 rHB84) duplication to ac42 r.7102 and selecting (r1589.r.7201) recombinants on a su- lambda lysogen (rHB84 is an A cistron amber mutation). These recombinants are then tested for their ac genotype (DOERMANN and BOEHNER 1970), from which the order of r1589 and rJ101 is deduced. The results of these crosses are presented in Figure 4. A mode containing 14 duplications exists at 0.2-0.3, while a second mode with 29 duplications occurs at 0.7-0.8. The former represents r.7102~1589 duplications and the latter r1589.r.7202 duplications. The excess of r1589.r.7201 duplications is significant (Chi' = 5.3, df = 1, P < 0.025) and has been previously observed by PARMA, INGRAHAM and SNYDER ( 1972). As expected, all 17 Class I11 duplications are in the upper mode; in Class I11 duplications only the

D. H . PARMA et al.

I

!1 I I I I

0 0.2 0.4 0.6 0.8 I .o PROPORTION of rHB84' RECOMBINANT

DUPLICATIONS TESTING ac+

FIGURE 4.-Results of Dp (r1589 . rJlO1 rHB84) acf x ac41 rJlOl crosses. Crosses of each original acriflavin-sensitive duplication to ac41 rJlO1 were performed in E . coli B as described for genetic mapping of duplication end points by PARMA and SNYDER (1973). For each cross the ac genotype of approximately 30 random plaques selected on K/S was determined by the procedure of DOERMANN and BOEHNER (1970). If rJlOl is in the right half of the duplication, the majority of these recombinants are expected to be acriflavin-sensitive, while if rJlO1 is in the left half, they will be predominantly resistant. The histogram indicates the proportion of (1-1589 . rJlOl rHB84 + ) recombinant duplications which are acriflavin-sensitive. The results for a particular duplication are indicated by its isolation number. The procedure is valid for duplications which include the ac gene, since the initial selection is for nmrHB84f recombinant duplications and is independent of ac genotype. During plaque growth on K/S duplications which are initially heterozygous ac+/ac41 will produce ac41/ac41 homozygotes by asymmetrical pairing and double crossovers. The presence of ac41/nc41 homozygotes in a plaque will cause it to test acr in the method of DOERMANN and BOEHNER. If the initial selection were for acr as well as amrHB84 $. , then the ac+/ac44 heterozygotes would be acriflavin-sensitive.

T4 rll DUPLICATIONS 599

r1589.rJIOI order allows both functional A and B products. Of the 22 Class I1 duplications in which the entire rZZ region is repeated, 13 are in the lower mode and nine are in the higher mode, indicating that either orientation is equally iikely and thus verifying the contention of PARMA. INGRAHAM and SNYDER (1 972) that the excess of r 1 5 8 9 ~ J I O I duplications is primarily due to Class I11 duplications. Further corroboration of the technique's reliability is that all nine duplications whose right-hand end points are in the A cistron are found in the 0.7- 0.8 mode.

Stability of duplications: The stability of each duplication was examined in single-cycle growth experiments by infecting CR ( A h ) at low multiplicity as described by PARMA, INGRAHAM and SNYDER (1972). A modification of the B:K plating technique of WEIL, TERZAGHI and CRASEMANN (1965) was used to determine tlie frequency of r segregants. Samples of each single-cycle growth lysate were adsorbed to E . coli B and the infected bacteria were plated on a 1:5 or 1: 10 mixture of CR/hh: CR(hh). Under these conditions segregants form turbid plaques and duplications form clear ones. A random sample of up to 100 turbid plaques was tested by the procedure of DOERMANN and BOEHNER (1970)

TABLE 2

Characteristics of tandem duplications in single-cycle growth

Class+

r1589 segre- Segregation gants/total frequency* r segregants Duplication Class+

Dp601 Dp602 Dp603 Dp604 Dp605 Dp606 Dp607 Dp608 Dp609 Dp610 Dp611 Dp612 Dp613 Dp614 Dp615 Dp617-2 Dp618 Dp619 Dp620 Dp621 Dp622 Dp623

I1 I1 I1 I1 I1 I I1 I11 I11 I11 I1 I11 I1 I

I11 I11 I1 I1 I1 I1 I1 I11

0.42 0.37 0.22 0.48 0.33 0.49 0.39 0.50 0.35 0.55 0.70 0.47 0.21 0.56 0.11 0.84 0.00842 0.002 0.11 0.70 0.21 0.46 0.0@49 0.00 0.21 0.84 0.52 0.58 0.47 0.93 0.030 0.00 0.21 0.58 0.24 0.51 0.23 0.20 0.22 0.44 0.232 0.332 0.27 0.87

Dp701 Dp702 Dp703 Dp705 Dp706 Dp707 Dp708 Dp710 Dp711 Dp713 Dp714 Dp715 Dp716 Dp717 Dp718 Dp719 Dp721 Dp722 Dp723 Dp725 Dp726

I1 I11 I

I1 I11 I11 I1

I11 I11 I1 I11 I11 I1 I1 I11 I1 I1 111 I1 I11 I1

r1589 segre- Segregation gantshotal frequency' r segregants

0.262 0.732 0.32 0.90 0.36 0.282 0.17 0.65 0.32 0.97 0.36 1.00 0.23 0.46 0.069 0.77 0.074 0.61 0.18 0.65 0.412 0.872 0.172 0.972 0.43 0.71 0.22 0.50 0.068 0.57 0.48 0.42 0.34 0.48 0.022 0.00 0.302 0.G2 0.202 1.00 0.21 0.78

* r segregants/total plaque-forming progeny. Superscripts indicate the number of independent

+ Class determined from acr mutation index and ac' triplication formation. experiments on which the ratio is based.


to determine the proportion of r1589 and rJ202 segregants. The results are presented in Table 2. The segregation frequencies range from 0.0049 to 0.697 and the r1589 proportion from zero to one. Duplications with the very low seg-regation frequencies exhibited by Dp609 and Dp612 have recently been reported by ROTHMAN, ROBBINS and LACKEY (1975). These low frequencies strongly suggest that only a very small portion of thy2 rZZ region is repeated in these duplications.

Mapping experiments employing point mutations: The mapping experiments in this section are fundamentally crosses between a nornial chromosome carrying a mutant allele ( a m ) and a duplication chromosome carrying the wild-type allele ( a m + ) . Duplications recombinant for the interval defined by the break- point and the mutant site will be heterozygous if the site is included in the dupli- catvzd segment (Le., all recombinants will still have one copy of the wild-type allele). i f the site is not included, they will be hemizygous for the mutant allele. Thus, it can be decided if the mutant site is included by the presence or absence of the wild-type allele in the diagnostic recombinant class.

Mapping right ends of Class ZZ duplications: Method A of PARMA and SNYDER (1973) was used; an rdP8~2589 derivative of each Class I1 duplication was con-

5- - ul $3- -

1 - 1 I I I I I I 5 IO 15 20 25 30

NUMBER OFDprdPB4r'RECOMBlNANTS TESTING AS WILD TYPE OUT OF 30

FIGURE 5.-Results of D p rdP8 . r1589 x mutant crosses. For each cross, 30 random progeny plaques on CR(1h) on acriflavin-supplemented plates were tested against the parental mutant. The number of wild-type tests obtained is recorded in the histogram. If no more than one mutant test was observed the site is considered to be included in the duplication. The result for a particular duplication is indicated by its isolation number. See PARMA and SNYDER (1973) for further details.

T4 rII DUPLICATIONS 60 1

strucied (see Figure 5, PARMA and SNYDER 1973) and crossed to a series of mutants in genes 60 and 39 (see Figure 6, PARMA and SNYDER 1973). Duplica- tions were initially crossed to E429, E300, E416, and E26. The results of these crosses are presented in Figure 5. Crosses to additional mutants were made to define more precisely the location of particular ends. PARMA and SNYDER (1973) considered a mutant site to be duplicated if they obtained not more than one mutant test in 30. However, their data were insufficient for a critical evaluation of the technique’s reliability.

The results for E429, E300 and E416 show a bimodal distribution, the upper and lower modes corresponding to duplicated and unduplicated sites respectively. The lower mode of the E429 and E300 distributions have a median value of four, while that of the E416 distribution is five. The upper modes contain values of 29 and 30. These data confirm the adequacy of the method and the convention of PARMA and SNYDER (1973). The absence of the upper mode in the distribution for E26 means that this site was not repeated in any Class I1 duplication.

Mapping right ends of Class I I I duplications: Method B of PARMA and SNYDER (1973), in which an r1589.r.7101 rHB84 duplication is crossed to an rJ101 gene- 60 or r.7101 gene-39 double mutant, was used to map the right ends of Class 111 duplications and of Dp622 (see Figure 7, PARMA and SNYDER 1973). Duplica- tions were routinely crossed to HL626, E416, NG604 and E205. Those duplications in which gene 60 is repeated were then crossed to additional mutants in gene 39 (E26, E556, E566) to define more precisely the location of ends. The results for HL626, E416, NG604 and E205 are presented in Figure 6. The results for HL626, E416 and NG604 each show a bimodal distribution, again the lower mode corresponding to unduplicated sites and the upper to duplicated ones. The median value of each of the lower modes is 11, which is significantly higher than those obtained by method A. This indicates that crossovers between the rII region and the mutant sites are appreciably more frequent in Method B than Method A. Whether this is an artifact due to difference in selection procedures, to a difference in the structure of the duplications, or to the repair of single- stranded deletion loops in in uiuo het-zroduplex DNA molecules (BENZ and BER- GER 1973) has not been determined.

While the resolution of Method B is not as good as that of Method A, the results with one exception are consistent with the convention of PARMA and SNYDER (1973). The one ambiguity, Dp725 tested against HL626, has been assigned to the upper mode since Dp725 gives 30J30 positive tests fo r both E416 and E26.

Initially the results for E205 are somewhat less satisfying. The lower mode corresponding to unduplicated sites has a medium value of 12 and Dp707 gives the expected result for a repetition of the E205 site. However, four duplications exhibit from 22 to 27 wild-type tests. This result is correlated with the duplication of the NG604 and E556 sites and suggests that the 39-rIZB hybrids have iimited functional capacities if the NG604 and E556 sites are included in the hybrid. However, the functional capabilities of such hybrid genes have not yet been examined.


t - 0 % g U Q

U U 0

a

LL 0

a .P w z z .z

r ( n, 1 1 , ~ , , , I

5 IO 15 20 25 30 NUMBER OF Dpr1589.rJIOI RECOMBINANTS TESTING WILD TYPE OUT OF 30

FIGURE 6.-Results of D p r1589.rJlOl rHB84 x rJlOl amber crosses. For each cross, 30 random plaques on CAJ (r1589.rJIOl rHB84C recombinants) were tested against the appropriate parental amber mutation. The number of wild-type (an+) tests obtained is recorded on the histogram. If no more than one amber test was observed, the site is considered to be duplicated. The results for a particular duplication are indicated by its isolation number.

Mapping ends in the A cistron: Method C of PARMA and SNYDER (1973), in which an (r1589.rJ101) derivative of the original duplication is crossed to rJ101 rHB118 to determine if the rHB118 site is repeated (rHB118 is a B-distal, A- cistron amber mutant), was used to analyze duplications not extending into gene 60 (see Figure 8, PARMA and SNYDER 1973). Recombinant duplications that are hemizygous o r homozygous for rHB118 form turbid plaques under the double- layer plating conditions of PARMA and SNYDER (1973). If the rHR118 site is ,duplicated, turbid plaques are rarely if ever observed, while if it is not duplicated, turbid plaques usually occur more frequently than in the rHB84 control cross. The results are presented in Table 3 and indicate that the right ends of Dp609, 612, 622, 703, 710, 711, 718, and 722 are in the A cistron between the B-distal end of r1589 and the rHB118 site.

Mapping experiments employing deletions: The mapping experim-ents in this section are basically crosses of a normal chromosome carrying a deletion to a triplication chromcsome that does not carry that deletion. If the site of the deletion’s left end is included in the duplicated segment, then recombinant triplications carrying the deletion in the right segment can be formed (PARMA and SNYDER 1972, Figure 10). Experimentally, the recombinant triplications are

T4 rZZ DUPLICATIONS

TABLE 3

Frequency of turbid plaques in Dp r1589.rJ101 x rJlOl rII amber crosses*

603

Frequency of turbid plaques Duplication Crossed to Crossed to HB118 site

parent rJlOl rHB84 rJIO1 rHBl l8 duplicated

Dp602 Dp606 Dp609 Dp610 Dp612 Dp622 Dp703 Dp705 Dp710 Dp711 Dp713 Dp718 Dp722

3.5 x 1 0 - 2 7.5 x 10-2 1.7 X 10-2

1.2 x 10-2

7.8 x 10-2 1.2x 10-2

2.8 X 1 0 - 2 1.2 x 1 0 - 2

8.7 x 10-4 4.2 x 10-2 6.5 x lor3 2.2 x 10-2

6.3 x 10-3

<3.8 x 10-4

3.1 X 10-2 <7.7 x lo"

1.6 X 3.5 x 10-2 3.1 X 10-2

<5.5 x lo" 2.4 X 10-2 1.7 X le2

1.6 X 1O-2 1.7 X

a . 8 x 10-3

5.1 x lor4

Yes Yes No Yes No No No Yes No No Yes No No

* Cross conditions are similar to those for end-point mapping methods A and B. Progeny are plated on CR(Vz) and 20 minutes later the plates are overlaid with K/S (plating bacteria diluted 102-fold) in 2 ml soft agar. After 20 min at room temperature the plates are incubated overnight. Under these conditions parental duplications make clear plaques while recombinant duplications, which are either hemizygous or homozygous for the rl l amber mutation, make turbid plaques. Recombinant duplications initially heterozygous for the amber mutation and its wild-type allele make clear plaques, since homozygous am+ duplications are generated by recombination during plaque growth on CR(hh), In those crosses in which no turbid plaques were detected, the frequency is indicated as less than the reciprocal of the total number of plaques accrued. The value for the Dp713 X rJlO1 rHB118 cross is based on one turbid plaque.

acriflavin-resistant. Thus, the presence or absence of acT triplications indicates whether or not a site is included in the duplication. This method is important because in the D region the only useful genetic markers x e deletion end points.

Mapping le f t ends of Class ZZ duplications: Method D' of PARMA and SNYDER (1973), which determines if the rdP8~1589.1589 triplications segregated from rdP8~1589 duplications will produce ac' triplications when crossed to the acriflavin-sensitive, B-cistron, B-terminal deletions r638, rPB296 and rNB3157, was used to further localize the D region ends of Class II duplications. Duplications that cannot form ac+ triplications in these crosses can produce acT duplications able to grow on a lambda lysogen by replacing rdP8 with a B-terminal deletion and mutating to acriflavin resistance. These phages are expected with the frequency of the duplication's acT mutation index, about

When crossed to r638, acriflavin-resistant plaques on CR ( A h ) were produced with frequencies ranging Prom to 3 x 10-I (data not shown). The results for crosses to rPB296 are shown in Figure 7. Only Dp605, Dp713 and Dp622 yield acriflavin-resistant plaques on CR (Ah) with a frequency significantly abore the mutation index. The results for the rNB3157 crosses are similar to those for rPB296 except that the frequency of plaques in the Dp622 cross was 2 x These results indicate that the left end of Dp605 and Dp713 is in the interval defined by the ac locus and the B-distal end of rNB3157, that the left end of Dp622


FIGURE 7.-Frequency of aer triplications in Dp rdP8 ,1589 x rPB296 crosses. The results for particular duplications are indicated by their isolation numbers. As expected, all crosses produced duplication progeny able to grow on CR(U). However, only in the Dp605, 622 and 713 crosses were acr triplications produced. The ratio of the figure is [progeny on CR(Ah) plus acriflavin]/[progeny on CRO\h)] rather than the ratio [progeny on CR(M) plus acriflavin]/[progeny on CR/Xh] used by PARMA and SNYDER (1973). This ratio was used since the crosses have only approximately equal parental inputs; it thus provides a more direct comparison with the frequency of ae? duplications (rPB296.rl589) generated by recombination and mutation to acriflavin resistance. The results for Dp622 are from a Dp rNB3162.1589 X rPB296 cross since Dp622 produces aer triplications in crosses to rNB3162 but not in those to rdP8 (see text).

is in the interval defined by the B-distal end points of rNB3157 and rPB296, and that the remaining Class I1 duplications all end in the interval between the B-distal end points of rPB296 and r638.

Mapping left ends of Class 111 duplications: The left end of Class I11 duplications can be localized between the A-distal border of the B cistron and r.7101, or tentatively between rJ101 and the A-proximal border, by segregation of r.7101 from the duplication (PARMA and SNYDER 1973). Table 2 indicates that r.7101 segregants have been recovered from all duplications except Dp707 and Dp725. A diagrammatic summary of mapping data thus far presented is given in Fig- ure 8.

Anomalous duplications: Results are summarized in Table 4 for duplications that behave atypically. The anomalies are of two types: (1) ambiguous experimental data; and (2) data not accounted for by a simple tandem duplication.

Dp617-1: The original stock of Dp617 contained duplications of two plaque types. One corresponds to Dp617-2, a very short but normally behaving Class I11 duplication. The other, Dp617-1, is nominally a Class I11 duplication. HOW- ever, the data from crosses to ac41 rJlO1 are ambigous, and the r.7101 segregation frequency seems unreasonably high for a Class I11 duplication. Dp617-1 may in reality be a Class I1 duplication that is too long to be compensated by rdP8. Verification of this explanation (which does not account for the ambiguity of order except that either order results in a functional duplication and the stock could be a mixture of both) depends on the isolation of ac' triplications.

T4 rl l DUPLICATIONS 605

I 703 I i 614 ! I 606

713 605 622 602,607,611,618,619,621,701,705,717,72l,723,726 708 60l,603,604,6l3,620,7l6,7l9 609,612,617-2,710,711,718,722 608,610 623,714 706 702,715 615 725 707

FIGURE &-Genetic map of duplicated segments. At the top of the figure is a genetic map of markers used in end-point mapping. All intervals are arbitrarily drawn with equal size. The duplicated segments are represented by horizontal lines which are drawn to the center of the intervals in which the end points occur. The rNB3157, rPB296,r638 and r1589 marks represent the left ends of the first three deletions and the right end of the fourth. A-B represents the A-B cistron divide, and A and B the borders of the A and B cistrons. Right ends are assigned to the A-E429 interval if the E429 site is not duplicated and the r H B l l 8 site is, or if the duplication forms UC' triplications in crosses to rdP8 or has the order rJlOl.rl589. The duplications corresponding to a particular segment are indicated by the isolation numbers to the right of the segment.

Experiments employing rdP8 in conjunction with a second nonessential deletion are in progress, as are additional ordering crosses.

Dp712: The ac" mutation index of Dp712 is variable. The rJ101 segregation frequency appears to be too high for a Class I11 and too low for a Class I duplication. Dp712 does not yield acr triplications when crossed to rNB3162 o r rdP8. The possibility remains that Dp712 is a Class I1 duplication with its right-hand end so close to r1589 that ac" triplications do not occur appreciably more frequently than do mutations to ac".

Dp720: The ac" mutation ratio of Dp720 is reproducibly 0.13. It appears that Dp720 is a Class I11 duplication that is especially sensitive to acriflavin, either because of its length or an unknown component of its genotype.

The remaining duplications, Dp616, 704 and 724, do not behave like simple tandem duplications. All three are atypically stable (Dp616 and 704 are extra- ordinarily stable for the segment duplicated) and produce rJ101 and r1589 segments in essentially equal numbers. Furthermore, in spot tests against r73, rJ101 and rJ87, their r1589 segregants invariably yield "wild-type" recombinants with rJ101 as well as r73, whereas the r1589 controls produce wild-type recombinants only with r73. This same pattern is produced by rdP8.i-1589 duplications segregated from acr triplications, indicating that the segregants still carry

606 D. H. PARMA et al. TABLE 4

Characteristics of anomalous duplications

Ratio of acr r1589 segre- mulation ac' Segregation gants+/total a d 1 rJl0It: Terminal interval

Duplication induces' triplications frequency r segregants order data of right ends

Dp617-I 0.32 <3.8 X 10-6 0.47 0.56 16/30 E26-E205 Dp712 variable 1.7 x 10-6 0.15 0.055 23/30 rl589-rHBll8

6.3 x 113-6 Dp720 0.13 1.0 x 10-6 0.374 1.00 27/34 E205- Dp616 0.57 2.3 x l o r 5 0.073 0.58 5/30 E566-E1204 Dp704 0.73 3.0 x 113-5 0.011 0.50 24/30 E556-E1204 Dp724 1.10 3.1 x 10-511 0.018 0.57 4/34 r1589-rHB118

2.4 x 10-3

* The ratio of (IC" mutation indices for the original Dp712 in four separate determinations were 0.099, 0.050, 0.046 and 0.097. For Dp616, 704, and 724 the ratio of acT mutation indices is not a valid test, since the segregants are homozygous duplications. However, the fact that both hommy- gous and heterozygous duplications and segregants have indices of about 10-5 indicate that the ac locus is not duplicated. + The r1589 segregants from tandem duplications and r1589 controls yield positive results when spot tested against r73 but negative results with rJlO1 and rJ87 [see Table 1 and DOERMANN and BOEHNER (1970)l. The r1589 segregants from Dp616, Dp704 and Dp724 invariably give positive results with rJ101 as do the rdP8.rI589 segregants from a@' triplications. Similarly the rJIO1 segregants from these three duplications give positive tests with r1589. These results indicate that all r segregants from Dp616,704 and 724 are homozygous duplications.

2 Ratio indicates the number of recombinant (r1589,rJIOI rHB84+) duplications which are acriflavin-sensitive out of 30 tested. The data for Dp617-I are ambiguous. Retesting this stock gave the result 8/30 indicating the order rJIOI.rl589; however, the results for two single-plaque subcultured stocks of Dp617-I were 14/30 and 13/30.

I/ Dp724 produces two plaque sizes when crossed to rdP8. Regular sized plaques occur with a frequency of 3 x 10-5 and are presumed to be duplications which have mutated to acriflavin resistance. Minute plaques occur much more frequently (2 x 10-3) but attempts to genetically analyze the contents of these plaques have been unsuccessful. However, this phenomenon is a genetic property of Dp724 since two single-plaque subculture stocks behave in an identical manner. The resolution of this ambiguity is currently being pursued.

Right-hand end points were determined by Methods B and C .

th.e duplication. Two hypotheses can account for these results: (1 ) an insertion duplication with essential DNA lying between the inserted region and the homologous normally located region; (2) a stabilized tandem duplication (VAN DE VATE and SYMONDS 1974). According to either hypothesis, only segregants produced by double crossovers are viable.

According to the insertion hypothesis, the repeated segment of Dp616 includes the entire rll region, all of gene 60 and a portion of gene 39, and r1589 is the inserted segment; the repeated segment of Dp704 includes all or part of the B cistron, the entire A cistron, gene 60 and part of gene 39, and rJlO1 is the inserted segment; the repeated segment of Dp724 includes the entire B cistron and part of the A cistron, and r1589 is in the inserted segment (Figure 9a-c). On the other hand, the stabilized tandem duplication hypothesis requires that the repeated segment of Dp616 includes the entire r l l region and all of genes 60 and 39, that r1589 is in the right half of the duplication, and that a stabilizing deletion penetrates the right half of the duplication ending between the E556 and E1204 sites; that the repeated segment of Dp704 includes all or part


B A 60 39 B A 60 39

(C) 1589 tc3)------ FIGURE 9.-Insertion hypothesis: structure of Dp616, Dp704 and Dp724. The normal r l l

region is represented at the left and the inserted region at the right. The dashed line represents unduplicated portions of the genome. Dp616,704 and 724 are depicted in a, b and c respectively.

of the B cistron, all of the A cistron, gene 60 and gene 39, that rJ102 is in the right half of the duplication, and that a stabilizing deletion penetrates the right half of the duplication ending between the E556 and E1204 sites; and that the repeated segment of Dp724 includes the entire A and B cistrons (and most likely all of genes 60 and 39) , that r1589 is in the right half of the duplication, and that a stabilizing deletion penetrates the right half of the duplication extending into the A cistron past the rHB118 site (Figure loa-c). While either hypothesis accounts for the data, they differ in their definition of the duplicated segment.

To confirm the adequacy of these hypotheses and further define the left end of Dp704, crosses were performed between complementing sibling segregants,

B A 60 39 B A 60 39 JlOl 1589

I 1 1 1 1 I l l , \ I I I \ I I ,

;s!

B A 60 39 JlOl I589 sd

I I I I I , 1 1 1 H (c) -I+ I *

'. :

60 39

FIGURE 10.-Stabilized tandem duplication hypothesis: structure of Dp616, 702 and 724. Tandem duplication structures for Dp616, 704 and 724 are depicted in a, b, and c respectively. In each case a stabilizing deletion, sd, is shown penetrating the right half of the duplicated segment. Note that not all sites in the duplicated segment (defined by the position of the join point) are present in the two copies. These structures are not uniquely defined by the data. For instance, the join point of Dp724 could be between the A cistron and gene 60. In this case the stabilizing deletion must not extend into gene 60 (d).


and between segregants and their complementing standard deletions. The frequency of functional (r.7101~1589) duplications among the progeny of each cross was determined by plating on CR ( A h ) . It can be shown that both hypotheses predict that if the entire rll region is duplicated, the complementary recombinants produced by an odd number of crossovers between the two rll regions are functional duplications and consequently their frequency is expected to be equal to the frequency of recombination for the interval separating r.7101 and r1589 (Figure I l a ) . When the 71589 r1589 segregant of such a duplication is crossed to a standard r.7101 deletion o r the r.7101.r.7101 segregant is crossed to a standard r1589 deletion, then only one of the crossover products will be a functional duplication, so that the expected frequency is half that in the sibling cross (Figure 1 Ib) . If the entire A cistron but only a portion of the B cistron is duplicated, then in the sibling crosses only one of the complementary recombinants generated by an odd number of crossovers between the 1.11 deletions is a functional duplication (the other is heterozygous but will have a truncated nonfunctional r1598 product when r1589 is in the right or inserted half) . Consequently, twice the frequency of functional duplications is expected to be equal to the recombination frequency for the interval separating the r l l deletions (Figure 12a). The expected frequency of functional duplications in the rJlOlv-.7101 segregant x standard r1589 cross is the same as in the sibling cross since once again, one of the complementary recombinants produced by

B A 60 39 B A 60 39

FIGURE 11 .-Formation of functional duplications in homozygous sibling segregant and homozygous segregant-X standard deletion crosses. A sibling segregant cross is represented in part (a). The duplication is depicted as an insertion with the entire rll region duplicated (see Figure 9) but exactly similar expectations can be derived for stabilized tandem duplications (Figure 10). The crossover praducts depicted by the solid and dotted lines are both functional duplications. Part (b) represents an r1589.rI589 x rJlO1 cross, rJlO1 being paired with the standard positioned rll region of the duplication. Only the dotted crossover product is a functional duplication. If rJlOl pairs with the inserted region in double crossover with one single exchange on either side of r1589 is required to produce a functional duplication, which will be relatively infrequent. Analogous predictions can be derived for r1589 x rJlOl. rJlO1 crosses.

T4 rZZ DUPLICATIONS 609

B A 60 39 B A 60 39

~, ............................................. -. .......

..... J,ol . ....-\ Jlo, U-+ _ _ _

+- - -- - - - - -

....................................

FIGURE 12.-Formation of functional duplications in homozygous sibling segregant and homozygous segregant-X standard deletion crosses. The entire A cistron but only a portion of the B cistron is included in the duplication depicted in Figure 12. In the cross between sibling segregants (a) only the recombinant represented as a solid line has both functional A and B cistrons. In the rJlOl~rJlO1 x r1589 cross (b) the dotted-line recombinant is functional. In the r1589-rl589 x rJlO1 cross (c) functional duplications are produced by rJlOl pairing with the inserted rll region and a double crossover, which is relatively rare.

an odd number of exchanges between the two rZZ deletions is a functional duplication (Figure 12b). However, in the r1589~1589 segregant x standard r.7101 cross, functional duplications are produced only by asymmetrical pairing and double crossouers, and the frequency of functional duplications is therefore expected to be much less than that in the sibling crosses (Figure 12c). If the entire B cistron and only a portion of the A cistron is duplicated, analogous reasoning predicts that the hequency of ftinctional duplications in the sibling and r-1589~1589 segregant x standard r.7101 crosses equals half of the recombination frequency between the rZZ deletions; and that the frequency in the rJ101~J101 segregant x standard r1589 cross is much lower. (In the context of this experiment, the duplicated segment is that region which i s actually present in two copies. Ordinarily the duplicated segment is that region defined by a duplication’s join point.)

The results of these crosses for segregants from Dp616, 704, 724, 615, and 726 (the latter two serving as normal tandem-duplication controls) are given in Table 5. The results for Dp704 are those expected for a duplication that includes the entire A and only a portion of the B cistron; while those for Dp724 are for a duplication of the zntire B, but only a portion of the A cistron. These

610 D. H. PARMA et al. TABLE 5

Resulis of crosses involving homozygous segregants*

Percent functional duplications among progeny in Sibling r1589 segregant rJ1Ol segregant

segregant X X Duplicated portion Duplication Series cross rJ1Ol standard r1589 standard of rll region

Dp616 1

Dp704 1

Dp724 1

Dp615 1

Dp726 1

2

2

2

2

2

7.4% 7.6% 1.3% 1.8% 2.2% 2.2%

<0.0099%

<0.0052% <0.01029%

<0.0077%

- - entire r l l 1.4% 0.85% - - entire A 0.028% 1.98% partial B - - entire B 1.9% 0.018% partial A - - not duplicated

- - not duplicated <0.0027% <0.0022%

<0.0032% < 0.0015 %

* Crosses were performed in CR(Xh) as described by PARMA, INGRAHAM and SNYDER (1972) for single-cycle growth experiments, except that the multiplicity of infection of each parental phage was 0.1. Total progeny were assayed on CR/hh and functional duplications on CR(Ah).

results are not only consistent with the previously deducted structures of Dp724 and Dp704, but also define more precisely the left end of the latter. The results for Dp616 depart from those expected for a repetition of the entire r l l region. Although the frequency of functional duplications in the r-1589~1589 x rJl01 cross is approximately equal to that in the r.7101 rJl0l X r1589 cross, their average value is only 1/6 that of the sibling cross (instead of 1/2). The cause of this departure from expectation has not yet been determined, but it could be due to a recessive mutation in the duplication genome that increases recombination or to complications associated with a compensating deletion. In any event, the results are deemed to be consistent with the proposed structures of Dp616.

The percent recombination between the rZZ regions of Dp616, of Dp704 and of Dp724 is 7.5%, 3.1% and 4.4Oj,, respxtively. The genetic distance between the two rll deletions is expected to differ under the alternative hypotheses. According to the stabilized tandem duplication hypothesis the distance is approximately equal to the length of the duplicated segment (as defined by the join point), while according to the insertion hypothesis it is approximately the length of the duplicated segment plus the unduplicated material between the two rZZ regions. Thus a minimum expectation for the frequency of functional duplications can be calculated from the genetic length of the repeated segment. For Dp616 and Dp704 the expected frequency is about 20% as deduced from the map sizes of the duplications and about 16% based o n r1.589 x E205 crosses. Since the ohserved frequ-mcies are less than this minimum, the results would seem to favor close linkage and therefore the stabilized tandem hypothesis. However, initial attempts to convert the duplications to normal tandem duplications by crossing r1589 r1589 segregants to rJlOl rHB84 r48 A33 and r.7101. rJlO1 segregants to r1589 r48 A 3 3 have not been successful. Consequently, a rigorous deterniinytion of the structures remains to bc made.

T4 rZZ DUPLICATIONS

TABLE 6

Results of r l W l x rJlOl crosses*

61 1

Cross Frequency of Frequency of

duplication plaques complementation plaques

r1231 x rJlOl 1.3 x 10-8 2.2 x 10-6 r1231 X rJ101 rHB84 r48 2.3 x 10-6 r1231 X rJlO1 rHB84 r48 A33 3.5 x 10-6

1.0 x 10-8

2.4 x l e 8

* Standard equal-input multiplicity crosses were performed in E. coli B according to CHASE and DOERMANN (1958) except that the multiplicity of each parent was 2. Total progeny were assayed on CR/kh. Duplication and complementation plaques were scored on CR(Xh) .

Frequency of duplication formation: Determination of the frequency of duplication progeny jn r.7101 x r1589 crosses is hiimpered by the presence of minute complcmentation hetct.ozygo te plaques which occur with frequencies in the range to In an effort to circumvent the problems created by these plaques, the occurrence of duplication progeny in 7-1231 x rJlO1 crosses has been examined. Like r1589, r1231 deletes adjacent portions of the A and B cistrons and overlaps r.7101. However, r1231 expresses B function only when “activated” by an A-cistron frameshift mutation (DRAKE 1963). Thus, in r1231 X r.7101 crosses the parental genotypes do not complement. In order to form functional duplications, the right end must occur in the r1231 A fragment and simultaneously shift the reading frame; the rZZ gene remnant must be attached to a promotor that functions at an appropriate time and rate; and the added N-terminal polypeptide fragment must not interfere with the B-cistron activity of the polypeptide chain. The subset of duplications isolated in these crosses is more restricted than, and in fact does not overlap with, those isolated from r1589 x r.7102 crosses. The results are presented in Table 6. The frequency of duplication plaques is about 1 x while that of complementation plaques is about 2 x Implications of these results will be considered in the DISCUSSION.

DISCUSSION

The present experiments were designed to examine relationships between duplication end points and to evaluate the effects of relaxed packaging restrictions on this distribution. Each duplication except Dp617-1 and Dp617-2 was isolated from a different selection plaque produced by plating mixed infected host bacteria before lysis. This is equivalent to isolating each duplication from a different cross. In all probability, viable duplications do not constitute a random sample of duplications involving the rZZ region since only those that produce adequate amounts of functional rZZ products are viable. For example, when a right break point is in the r1589 A cistron, the break point must not cause a reading frame shift. Thus, in this subclass of duplications, as many as two- thirds of the end-point combinations may be lost. While sampling errors due to frame-shift restrictions are presumably not operative for break points outside the A cistron, regulatory requirements (i .e. , the requirement that the rZZ gene


in the left half of a duplication be attached to a promotor that functions at an appropriate time and rate) are still in effect, Another sampling error that may exist is the relatively inefficient recovery of longer duplications due (1 ) to the requirement for a long compensating deletion and (2) to their greater instability.

The requirement for a compensating deletion arises from the “headful” packaging system of T4. To assess the effects of relaxed packaging restrictions, parents of the 600 series duplications contained the 4000 base-pair deletion A33. As expected, the presence of A33 increased the frequency with which viable duplications were detected amongst the cross progeny. The increment was a factor of ten compared to the A33+ control (the 700 series). The effects of A33 can thus be evaluated by comparing the results of the 600 and 700 series. The numbers of Class I, I1 and I11 duplications provide a comparison of left end distributions. There are two, 13, and seven for the 600 series, and one, ten, and ten for the 700 series, respectively. The distributions are not significantly different. A similar comparison for right ends can be made using the following intervals: within the A cistron, between the A cistron and gene 60, within gene 60, between genes 60 and 39, within gene 39, and beyond gene 39 . The numbers observed for the 600 series are 4, 8, 1, 7, 1, and 1, while for the 700 series they are 5, 7, 1, 3, 4, and 1. Again the distributions are not significantly different. (Except as noted, the six anomalous duplications are excluded from the DISCUSSION. However, their inclusion would not alter the conclusions.)

At the level of discrimination provided by these data, A33 has no effect on the distribution of ends. This must mean that A33 is long enough to compensate most of the rZZ duplications that are ordinarily viable and that the average compensating deletion need be no longer than about 4000 base pairs. Measuring the physical size of the duplications under study and their compensating deletions is the most direct way of testing this explanation. This has not yet been done. However, the data of HOMYK and WEIL (1974) bear on the average size of compensating deletions. The 23 compensating deletions they studied ranged in size from 4,200 to 9,800 base pairs. The median length was 4,956 base pairs, with only five longer than 6,000 and only two longer than 7,000 base pairs. Thus while ~ 3 3 is smaller than any deletion characterized by HOMYK and WEIL, it is nonetheless near the mean size of those deletions.

The similarity of the 600 and 700 series’ distributions of ends allows pooling the data for further examination. The right ends of the duplications are distrib- uted as follows for the six intervals previously defined: Class 11: 1, 13, 2, 7, 0, and 0; Class 111: 7, 2, 0, 2, 5, and 1. While the data are not extensive, the distributions show significant differences; 22 of 23 Class I1 right ends occur in the region bounder by the A cistron and gene 39, while only four of 17 Class I11 right ends terminate in this interval (x2 = 22.0, df = 1, P < 0.01). Class III’s on the other hand are very likely to end within the A cistron or within gene 39 (12 of 17 or 71%), whereas only one of 23 Class II’s end in these genes. While the absence of Class I1 ends in gene 39 could be attributed to size-associ-

T4 rll DUPLICATIONS 613

ated difficulties, such is not the case for Class I1 ends in the A cistron and Class I11 ends in the A cistron-gene 39 interval.

Similar comparison can be made between those duplications that include the entir2 A cistron and those including only a portion thereof. If the right end point occurs in the A cistron, the left end is most apt to be in the B cistron (seven of nine or 78%), while only 27% of those duplications whose right end point is beyond the A cistron have left end points in the B cistron. Thus, at the cistron level of genetic organization, there appears to be a nonrandom relationship between the position of left and right ends of viable tandem duplications. However, the factor (s) determining such nonrandomness remain to be iden- tified.

If nucleotide sequence homology determines the position of ends, the size of identical sequences can be approximated from the frequency of end-point sites. For left ends, there is a minimum of five end-point sites in the D region plus the B cistron. If it is assumed that each corresponds to a different sequence, then each is occurring with a frequency of about l/5000 (BUJARD, MAZAITIS and BAUTZ 1970). On a random basis, a sequence 6-7 nucleotides long occurs with this frequency (this calculation is an approximation neglecting the A-T bias in T4 DNA). If the five sites are assumed to have the samre sequence, then the size is five. Similar calculations for right ends yield similar estimates.

Knowledge of the amino acid sequence OP the A, B and gene-39 proteins would be instructive in further determining the role of nucleotide sequence in the formation of break points. Tentatively, one may conclude that if homologous sequences are involved, they are short; they are of the order of six base pairs if identical, while somewhat longer if nonidentical. EMMONS and THOMAS (1975) have concluded that at most little sequence homology is necessary for duplication formation in phage A. In any event, the sequences would seem to be much shorter than T4’s “homologous pairing region” for recombination (DRAKE 1967).

PARMA and SNYDER (1973) observed a direct relationship between duplication size and segregation frequency. Implicit in their analysis was the assumption that duplication strains are isogenic except for the repeated segment. However, their strains and those under consideration here can depart from isogenicity in at least three ways: (1) loss of different genes due to different compensating deletions; ( 2 ) different terminal repetition lengths due to variation in the relative size of duplications and their compensating deletions; (3) effects of duplicated genes. Various departures of each type can lead to a segregation frequency that is lower or higher than normal for the segment size involved. Furthermore, when triplication segregants are inviable, the segregation frequency must be corrected to render it equivalent to recombination frequencies (see Figure 1 of PARMA, INGRAHM and SNYDER 1972). If triplications are as frequent as r segregants, as suggested by the data of PARMA, INGRAHAM and SNYDER (1972), the Corrected segregation frequency is twice [ r segregants/(duplications + 2{r segregants}) ] (PARMA and SNYDER 1973).


0 0

56-

> 0

3 0 W E

z

248-

4 0 0

g 3 2 c3 w v,

I- o w E

o

9 24.

8 16

6 8.

c-- c

-am , 1 I I 1 5 -

I I , , 20 I 39 40, I , 50 IO r1589 rAP129 iE300; E26E566 'E5t6 E205

E429 E416 NG60h El204 GENETIC MAP LENGTH

FIGURE 13.-Corrected segregation frequency versus genetic map length. The corrected seg- . . . . .... . .~ . . . . - - . . - ~ . .

regation frequency of the rl l deletion in the left half of a duplication multiplied by 200 (which is the equivalent to percent recombination) is plotted against the genetic map length of the interval in which the segregant-producing crossovers occur [i.e., the right end of r1589 to the right side of the join point; see Figure 1 of PARMA, INGRAHAM and SNYDER (1972)]. Corrected frequencies for duplications with the same right terminus are averaged (except those ending within the A cistron). Vertical arrows depict one standard deviation of the averaged frequencies. Results for the 600 and 700 series are represented by 0 and respectively. The genetic map length is that of BARRICELLI and WOLFE (1965). The dashed line represents the relationship of percent recombination to genetic map length. The solid line is the set of points where percent recombination equals map length (i .e. , complete positive interference).

The corrected segregation frequencies for left-hand markers are plotted against genetic map length in Figure 13. The data are quite variable. However, a number of qualitative conclusions are evident. In general, there is a direct relationship between genetic length and corrected segregation frequency. Exceptions do occur and are probably due to lack of isogenicity. Practically, this means that some caution must be exercised in estimating duplication length from segregation data alone. Segregation occurs 3-4 times more frequently than recombination for the same interval. This observation in itself does not meilii that crossing over is more frequent in duplications. It could be a reflection of altered interference par- ameters. However, the fact that the points tend to plot above the line where recom-


bination frequency equals map distance indicates either that there is more crossing over in duplications than in standard crosses or that the Barricelli-Wolfe mapping function underestimates the number of crossovers in T4. The latter is plausible since the mapping function of STAHL, EDGAR and STEINBERG (1964) gives significantly greater estimates of map distance.

A second set of data that bears on the recombination problem is the frequency of functional duplication progeny in sibling segregant crosses of the presumed stabilized tandem duplications Dp616 and Dp704. The duplicated segment in Dp616 includes the entire rll region, gene 60 and gene 39, while that of Dp704 includes part of the B cistron, the entire A cistron, gene 60 and gene 39. An expected frequency of functional duplications can be calculated from the genetic map length of the duplicated segment. The expected frequency is about 20%; the observed frequencies are 7.5% for Dp616 and 3.1% for Dp704.

These crosses differ from segregation experiments in three important respects: (1) segregation is due to assymmetrical pairing whereas iormation of functional duplications is via symmetrical pairing; (2) r segregants but not functional duplications can be produced by matings between sister replicas; ( 3 ) r segregants but not functional duplications can be produced by intramolecular crossing over.

Preferred asymmetrical pairing seems to be an inadequate explanation for high segregation frequency. In a random-mating system, symmetrical and asymmetrical pairing are expected to be equally frequent. Thus one-half of the matings could lead to segregation, just as one-half of the matings (those between molecules of opposite parental types) are potentially recombinogenic in standard mapping crosses. If the approximately 5-fold lower-than-expected frequency of functional duplications is due to preferential asymmetrical pairing, then these latter account for 90% rather than 50% of the pairings. The resulting increment in segregation frequency would be only a factor of 1.8. In any event, the maxi- mum increment is two-fold. Intramolecular crossing over also appears to be inadequate. The data of PARMA and INGRAHAM (1970) and PARMA. INGRAHAM and SNYDER (1972) suggest that tandem duplications produce triplications and T segregants with equal frequency, implying that intramolecular recombination is relatively rare. However, their experimental technique is not sufficiently sensitive to make the conclusion compelling. BELLETT, BUSSE and BALDWIN (1971) have unequivocally shown that duplications in phage h usually segregate triplications and unduplicated chromosomes in equal numbers. While these data are in a different system, they strongly support the notion that intramolecular crossing over is relatively rare in phage systems.

The fundament21 observations that require explanation are (1) that asymmetrical pairing seems to be more frequent than symmetrical pairing and (2) that there seems to be a reduction in the number of symmetrical pairings. Preferential asymmetrical pairing partially satisfies these requirements but does not allow for a sufficient increase in the number of asymmetrical pairings. Furthermore, a formal model to generate preferential asymmetrical pairing would have to be of the following kind. An initial association of molecules, which is not homology dependent, occurs and involves sequences larger than the size of the duplications


under study. Once this association occurs the two molecules move randomly in linear rather than in three dimensional space with respect to each other. There is a relatively high probability that upon resching the first region of homology, pairing for recombination will occur, the homologous pairing region being small compared to the duplications studied (DRAKE 1967).

A way of reducing the proportion of matings which are potentially recombinogenic symmetricals is by assuming two populations of randomly mating molecules. One population consists of sister replicas and the other of nonsisters. In standard and sibling crosses, matings between sister replicas are not potentially recombinogenic. However, in segregation experiments asymmetrical but not symmetrical pairings involving sister replicas are potentially recombinogenic. If the frequency of sister replica matings is assumed to be high, then there is apparent preferential asymmetrical pairing and a sufficient increase in the number of such ratings to account for the high frequency of segregation. How- ever, additional assumptions must be made to explain the lower number of symmetrical matings.

Since neither alternative seems adequate, they are not mutually exclusive, and other explanations are not ruled out, more data on recombination in duplications are essential to productive model building.

DRAKE (1967) has estimated the size of the pairing region in T4 to be between 100 and 3,000 base pairs. While our data are consistent with this estimate, they suggest that the upper limit is not more than 1,300 base pairs. The segregation frequency per genetic map unit is expected to decrease dramatically for duplications that are smaller than the size of the pairing region. In the Class 111 duplications whose right ends in the r1589-rHB118 interval, the number of bases avail- able for pairing is less than the rZZ region minus the length of r.7101, r1589 and the AI-A2h3bl segment, or about 1,300 base pairs (BUJARD, MAZAITIS and BAUTZ 1970). In these duplications r1589 segregants are produced by crossovers in the region defined by r1589 and the right end, which is necessarily shorter than the r1589-rHB118 interval. Crosses between r1589 and rHB118 produce about 2.9% recombinants, while Dp710, 711 and 718 produce lo%, 8.4% and 7.2% r1589 segregants respectively. Since these values are 2.5-3.4 times as frequent as normal recombination, they suggest that the duplications are larger than the homologous pairing region. Further precision of the pairing region’s size can be expected from more exact end point mapping of those Class I11 duplications whose right ends are in the A cistron.

An accurate estimate of the Irequency with which duplications occur is de- sirable because duplications are considered to be raw material for evolution. Estimation of the frequency of duplication formation in T4 from the frequency of rZZ duplications is theoretically and technically difficult, perhaps impossible. First, only those duplications that produce adequate amounts of functional r l l product are viable. Second, only relatively short duplications are recover.zd due to the need for a compensating deletion and the instability of large duplications. Third, the requirement for a compensating deletion makes the formation of viable duplications a two-step process. Fourth, the enumeration and identification of

T4 r l l DUPLICATIONS 61 7

duplication plaques on selection plates is rendered difficult by the presence of minute complementation heterozygote plaques, which occur orders of magnitude more frequently, and by the variation in duplication plaque size on selection plates. Fifth, the extreme variation in duplication plaque size on selection plates (from minute to wild type) and the facts that the selection plate plaque size i s not a genetic property and that functional duplications reconstructed by normal recombination in rdP8~1589 x r.7101 and rJlOl.rJ101 x r1589~1589 crosses have the normal plaque size for that duplication suggest that one or both steps of viable duplication formation occur on the selection plate. That viable duplication formation does occur on selection plates can be demonstrated by plating the parental mixture at concentrations like that of the progeny (PARMA, INGRA- HAM and SNYDER 1972).

Attempts to improve the system to yield a better estimate of duplication formation frequency have been of two kinds: ( 1 ) inclusion of a long nonessential deletion in the parents to convert the formation of viable duplications to a one- step process; and (2) use of r1231 in places of r1589 to eliminate complementation between parental types. Both modifications are somewhat successful. Viable2 duplications are recovered more frequently when a homozygous deletion is included in the crosses. and complementation plaques occur with a frequency of only 2 X loL6 in r1231 X rJ101 crosses, allowing much better enumeration of duplication plaques. In the latter crosses the appearance of complementation plaques was initially surprising. In restrospect they may result from either of two conditions: (1) cells infected with rJlO1 and a frameshift-activated r1231 (either as terminal redundancy heterozygotes or as mixed infections) ; and (2) cells infected with an uncompensated duplication and a complete genome’s information( either due to mixed infection or infection by a physiological variant of larger than normal head siw) . Initial plating experiments indicate that the minute plaques are initiated by more than one phage particle, thus favoring the mixed infection alternatives.

A working hypothesis can be formulated from the foregoing observations. Usually duplication genomes do not initially contain a compensating deletion. However, under standard selection procedure, they mixedly infect a bacterium with a nonduplication phage and grow for a number of infectious cycles as a complementation plaque. During these cycles a compensating deletion may arise, after which time the duplication will grow on single infection. Thus, the size of the original duplication plaque is a reflection of the time at which the compensating deletion occurred and is not a genetic property of the phage. As an initial test of this hypothesis, experiments are in progress to determine (1) the exact genotypes of the phages in minute plaques (specifically, whether the r1231’s are frameshift-activated) ; and (2) the number of particles required to initiate a duplication plaque.

The occurrence of deletions during growth as a complementation plaque may also account for the phenomenon reported by WEIL, TERZAGHI and CRASEMANN (1965) and SYMONDS et al. (1972) that apparently more than one duplication can be isolated from some selection plaques. Their conclusion is drawn solely


from differences in segregation frequencies, and under their selective conditions all duplication plaques are extensively contaminated by complementation plaques. Nonetheless, if a deletion spans the join point of the two halves of a duplication, then a shorter but normal tandem duplication with a new join point is generated. Two other types of deletions may increase the stability of tandem duplications without altering the repeated segment (i.e., without forming a new join point) : (1) a deletion contained entirely within one-half of the duplication; (2) a deletion that begins outside the duplication and penetrates one-half. I n each case the shorter or modified duplication can be expected to have a selective ad- vantage over the original one. Thus, detailed mapping of apparently different duplications isolated from the same selection plaque may be instructive.

A minimum estimate of the lrequency of duplication iormation per genome can be calculated from the r1231 x r.7101 cross data. Only the Ala through A2h3bl segments or the A cistron are not deleted by r1231. This interval contains 34% of the A cistron mutational sites and is therefore estimated to be about 970 base pairs in length (BENZER and CHAMPE 1961; O’FARRELL, HUANG and GOLD 1973). If it is assumed that this is a representative segment of the 166 kilobase- pair T4 genome, and that frameshifts anywhere in the interval will activate r1231 [this is probably not true since DRAKE’S (1963) suppressors all map in the A2h3 segment], then a minimum estimate of the frequency of duplications per genome is 1.7 X or 170 times that of r1231 duplications. If the complementation plaques prove to be uncompensated duplications, then this miniurnum estimate could be as high as 3.4 x lo4. If join points are restricted to the A2h3 segment, both estimates must be increased ten-fold. On the other hand, if the complementation plaques are largely due to mixed infections involving activated ~-1231’s~ then the significance of any frequency is in doubt since there are numer- ous rounds of growth on the plate in which duplications themselves can be formed.

Detailed genetic analyses of 58 duplications indicate that at least 55 are tandem duplications. The remaining three are presumed to be stabilized tandem duplications, but the possibility that one or more of them is an insertion duplication has not been definitely eliminated. Since insertion duplications in principal are compatible with the selection technique, the failure to recover them indicates they are rare, as might be expected due to the requirement for two join points.

This research was supported by National Science Foundation Grant No. GB-22611 and Public Health Service Grant No AI-09943. The senior author wishes to thank Dr. S. S. Hsu and Mr. DEAN W. SHELTON for their expert technical assistance.

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DISTRIBUTION AND FREQUENCY OF THE rZZ REGION … · 2003. 7. 26. · DISTRIBUTION AND FREQUENCY OF TANDEM DUPLICATIONS OF THE rZZ REGION OF BACTERIOPHAGE T4D DAVID H. PARMA, G. THOMAS