THE GENETIC CONSTITUTION OF TANDEM ... - Genetics

THE GENETIC CONSTITUTION OF TANDEM DUPLICATIONS OF THE rII OF BACTERIOPHAGE T4D DAVID H. PARMA

AND

MARLENE SNYDER

Department of Biology, University of Utah, Salt Lake City, Utah 84112 Manuscript received August 31, 1972 Revised copy received October 30, 1972 Transmitted by F. W. STAHL ABSTRACT

Methods for genetically mapping the end points of tandem -1plicaticEns c the rII region of bacteriophage T4D are presented. Analysis of ten duplications indicates (1) that the position of duplication end points and therefore the length of the duplicated segment differ in strains of independent origin; (2) that there is a direct relationship between segregation frequency and length; (3) that segregation is more frequent than expected on the basis of standard genetic mapping; and (4)that while duplications frequently include non-rII genetic material, frequently they do not include the entire rII region. The duplications studied range from less than two to about five cistrons in length.

a cross between the complementary, overlapping rII deletions rJlOl and '?589, exceptional progeny arise which are able to grow on bacteria lysogenic for phage X (WEIL,TERZAGHI and CRASEMANN1965). The principal characteristics of these exceptional phages, reported by WEIL,TERZAGHI and CRASEMANN (1965) are (1) each exceptional phage produces three types of plaque-forming progeny: exceptional phage like itself, rJIOl progeny and 7-1589 progeny; (2) under defined conditions each independently isolated line produces the three types of progeny with a characteristic frequency; (3) as a rule, the r progeny ( r segregants) are hemizygous rather than homozygous; and (4) in single cycle growth, the kinetics of r segregation and the r segreganl clone size distribution resemble those of recombination. To account for these observations, we have proposed (PARMA and INGRAHAM 1970; PARMA, INGRAHAM and SNYDER1972) that the exceptional phage contains a tandem duplication for a portion of the T4 genome that includes the rII region and that the length of the duplication varies from one isolate to another. By analogy to higher organisms, the tandem duplications were presumed to undergo both symmetrical and asymmetrical pairing. A similar hypothesis has recently been proposed by SYMONDS et al. ( 1972). An asymmetrically paired tandem duplication of the rII region is presented in Figure 1. A phage carrying such a duplication is in a general sense partially diploid. The recombinations depicted by dashed lines x and y produce hemizygous rJIOl and r1589 segregants, respectively. The r segregants are produced by recombination and therefore have recombinant kinetics and clonality. T h e relative and absolute frequency of rJlOl and A589 segregants is a function of the size of Genetics 73: 161-183 February, 1973.

162

D. H. PARMA A N D M. SNYDER

LEFT B A 8262 I

Y

x

~ 4 l I

RIGHT B A rJlOl

-r1589

-

....... . ..+. . . ............ Il a

I nl

I

I I

s2

E51

t

I

I

----------k...(................. -----I . .*...............4E. L---- -- r - - \ \ \ \

FIGURE 1 .-An asymmetrically paired tandem duplication of the rII region of bacteriophage T4D. The duplication's break point is represented by the squiggly vertical line. The left end point is between m41 and the B cistron and the right between the A cistron and S2. In this and other figures, the order r1589.rJ101 is assumed because it is more frequent among our duplications.

the bracketed regions x and y. In a test of the hypothesis, three predictions were INGRAHAM and SNYDER1972). First, duplications segregate verified (PARMA, triplications (Figure 1, dotted lines). Second, triplications occur with the same frequency as structurally normal chromosomes among the progeny of duplications. Third, the r mutation in the left half segregates by recombination to its right and the r mutation in the right half by recombination to its left. It should be noted that because of the restriction on the amount of DNA that can be packaged into a T4 head, phage particles containing a triplication segregated from a duplication lack plaque-forming ability, but they can complement amber mutants in a helper phage assay and can thus be titered. It is possible to generate viable (plaque-forming) triplications, which carry three distinguishable overlapping deletions, by recombination between triplications segregated from duplications and a long acriflavin-resistant r l l deletion which has neither A nor B cistron function (PARMA, INGRAHAM and SNYDER1972 and Figure 3 ) . These recombinant triplications are selected by their ability to form plaques on a A lysogen in the presence of acnflavin and are termed acriflavin-resistant (acr) triplications. This communication presents methods for genetically mapping the end points of duplicated segments and the results of a critical test, based on these techniques, of the assumption that the length of the duplicated segment differs in strains of independent origin. The results show (1) that the position of duplication end

TANDEM DUPLICATIONS

163

points and therefore the length of the duplicated segment differ in strains of independent origin; (2) that there is a direct relationship between segregation frequency and length; and (3) that while duplications frequently include nonrII genetic material, frequently they do not include the entire rII region. MATERIALS A N D M E T H O D S

Phage strains: Mutations of bacteriophage T4 used in these experiments are given in Table I. Their relative map positions are indicated in Figure 2. Unless noted, all mutants originated in TABLE 1 Phenotype and gene number of mutants Gene number

Mutation

amber

ac

acriflavin-resistant

ac-rII

D region-rIIB rIIB rIIB-rIIA

rIIA

E26 E52 E566 E556 E1204 NG604 E205 S2

’

Phenotype

52

acriflavin-resistant-B terminal deletion; isolated in T4B; deletes entire r11 region acriflavin-resistant-Bterminal deletion; deletes all of B and most 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 umber; rB94 site is deleted by rJl01; isolated in T4B small deletion; overlaps r1589 A-B deletion with B function; isolated in T4B small deletion, covered by rl589 amber; rHB84 site is deleted by r1589; isolated in T4B point mutation umber; isolated in T4B point mutation point mutation; isolated in T4B

60

smber

39

amber

38

amber

164 H

38

D. H. PARMA A N D M . SNYDER M

52

H

I

rXIB

.

-r73

E663

60

rZTA

iHBll8

1HB64

39

I

1AP129 E300 E26 E556 E205 ~219 E429 E416 E566 E1204

170

1 .

I

\ '

1

I

'

l-3.I-+3.3+2D+2.0+42~2.l~+3B~+I.~+3.0i

k124 +39-

+132+8.2---1

-3.5+5584

+8

11589

+8.5

MB [NE2226

. 6 L 4 . I 2 - l ______(

+7.03+

H 1.9

I

,+

8.12I-.

+4 . 1 4 -5.22

tN83157 I

FIGURE 2.-Genetic map of the gene 38 to gene 60 region. Intervals are not drawn proportional to genetic size. The order of left ends of deletions rNB3157, rPB296 and r638 is from BAUTZand BAIJTZ (1967) and DOVE(1968). Markers enclosed in parentheses are not ordered with certainty. HL626 and E429 map at or near the same site as do E52 and E26. Recombination percent is twice the percent wild-type recombinants determined by selective plating. Superscripts indicate the number of times a particular cross was performed. strain T4D. T4B mutants, with the exception of r638, rPB296 and rNB3157, were cross-reactivated into T4D as described by PARMA,INGRAHAM and SNYDER(1972). Mutant phages were S. P. CHAMPE,A. H. DOERMANN and W. B. WOOD. obtained from H. BERNSTEIN, Bacterial strains: Relevant properties of several E. coli strains are in Table 2. In addition, CR63/s, B/s, K112-12(h) #3/s (DOERMANN and BOEHNER 1970) were used as testing bacteria in the determination of phage genotypes. E. coli B and 011' served as hosts for crosses. Plating and culture conditions: Hershey's nutrient broth (CHASEand DOERMANN 1958) was TABLE 2

Properties -Bacterial Spain CR63/Ah

Symbol

of

bacterial strains Relevant properties

CR/Ah

permissive for all phage genotypes

CAJ70

CAJ

opal suppressor, A lysogen; used as permissive host for NG604 and for gene 39, 52 and "E" ambers except E429, E300 and E52 because of larger plaque size and higher efficiency of plating; restrictive for rII ambers

S/6

S/6

restrictive for ambers; used as host in testing leaky ambers from genes 60, 39 and 52; and for selecting wild-type recombinants in amber x amber crosses

CR63 (Ah)

CWh)

permissive for all genotypes except non-amber rII mutants

K12 (A) /s

K/s

01I'

011'

streptomycin resistant, X lysogen; restrictive for amber and rII mutants; used in testing rII amber mutants and for selecting wild-type recombinants in rII x rII and rII x amber crosses Su,, derivative of E. coli B; used as host bacterium in amber x amber crosses

165

T A N D E M DUPLICATIONS

TABLE 3

Test conditions for identifying amber mutants in genes 39,52,and 60 Mutation

Titer of phage suspension

Test bactexium

1 x 108

S/6 plating bacteria

35°C

1 x 108

S/6 plating bacteria

room temperature

none

S/6 plating bacteria

room temperature

Incubation temperature

E429 HL626

E1217 E26 NG604. E1204 I E663 J E916 E205 E556

1

used throughout. Bottom agar and top agar were prepared as described by CHASEand DOERMA“ (1958). Exponential plating cultures were prepared according to EPSTEIN(1958). Incubation of plates for phage assays was normally overnight at 35”C, but in some cases, as noted, was at mom temperature. Acriflavin-supplemented plates contained 2 pg acriflavin neutral/ml of bottom agar. Genetic mapping crosses: Equal input multiplicity crosses were performed according t~ the procedure of CHASEand DOERMANN (1958). The criteria of acceptability of DOERMANN and HILL (1953) were used. The recombination percent equals two times the percent of wild-type recombinants determined by selective plating. In crosses between non-rII ambers, wild-type recombinants were selected on S/6; in those between 7-11 mutants o r rII and non-rII ambers, selection for wildtype recombinants was on K/s. Genetic mapping of duplication end points: Nominally equal input crosses, based on total plaque-forming titers, were performed as described for genetic mapping. However, the criteria of DOERMANN and HILL(1953) for acceptability were not stringently applied. First, the duplication parent in each cross is a mixture of normal, duplication and triplication chromosomes, rendering obscure the meaning of equal input. Second, only intracross results, which are relatively insensitive to changes in multiplicity of infection, are used. The different modifications of end point mapping are described in detail in the text. Determnation of genotype: The procedure of DOERMANN and BOEHNER(1970), was used. Tests for rII amber mutations used a 50:l mixture of K/s and B/s. Certain amber mutants could not be successfully tested on B/s and CR/s strains. For these mutants, the conditions in Table 3 proved adequate. Nomenclature: Duplication strains are indicated by the symbol D p and an isolation numberThe term “normal chromosome” means one which does not carry a duplication. A “viable” phage particle is one which has “plaque-forming ability,” while an “inviable” particle lacks plaqueforming ability but can complement amber functions in a helper phage assay. The left to right map order used her corresponds to the clockwise order of the T 4 genetic map. RESULTS

Operationally, tandem duplications of the rII region can be divided into three group; on the basis of the position of their left-hand end point: those in which it is to the rII-distal side of the ac locus such that the ac locus is duplicated, class I (Figure 3a) ; those in which it is between the ac locus and the rII B cistron such that the entire B cistron is duplicated. class I1 (Figure 3b) ; and those in which

-

166

D. H. PARMA A N D M. SNYDER

(0)

a+

act

11589

(

-rJlOl D

A

(b)

act I

B

-rJlOl -r 1589 : I Jt; B e

A

s2+

I -

I

A

s 2+ I

U D

I 1

A

FIGURE 3.-Operational classes of rII tandem duplications. it occurs within the B cistron such that only a portion of the B cistron is duplicated, class I11 (Figure 3c). This operational classification is based on a duplication's mutation index to acriflavin resistance and on its ability to form acriflavinresistant (ac.) triplications. Acriflavin resistance is recessive to sensitivity. Thus, a phage carrying two copies of the ac+ gene must undergo two mutational events in order to grow in the presence of acriflavin. Duplications of class I (Figure 3a) show an abnormally low mutation index to uc' since they carry two ac+ loci, while types I1 and I11 will show normal indices (Figures 3b and 3c). It has previously been shown that in crosses of an ac'-rII deletion, such as rdP8, to an acriflavin-sensitive duplication it is possible to select plaque-forming ac' triplications which arise by recombination between rdP8 and triplications segregated from the duplication (Figure 4 and PARMA, INGRAHAM and SNYDER 1972). Since acriflavin resistance is recessive, the formation of ac' triplications is possible

P8

LJ 101

I:

(

I

a a

I

t

FIGURE 4.-Formation of an nc*-triplicationby recombination between rdP8 and the left-hand triplication. The crossover depicted by the dashed line yields an segment of an r1589~r1589~rJ101 ac* triplication.

167

TANDEM DUPLICATIONS

TABLE 4 Criteria for the operational classification of rII tandem duplications* Duplication class

I I1

I11

acr mutation

index

low normal normal

acp triplicatim formation

no Yes no

* The normal acriflavin resistance mutation index is about 10-5.

only if the repeated segment does not include the ac locus, since the rdP8.ac+rl589-ac+rJlOl (or rdP8 ac+rJI 01 .ac+rl589) triplications still carry two ac+ genes and hence are sensitive. Thus class I duplications cannot form acr triplications. In duplications in which the ac locus is not included, rdP8 can be recombined only into the left-most segment (because the site of the deletion's left end is not included in the duplicated segment). When rdP8 is recombined into the left-most segment of a triplication in which the left and center segments carry the same functional cistron and the right segment the other (i.e., r1589~r1589.rJ101 or rJ101.rJ101 ~ 1 5 8 9 the ) ~ recombinant is acr and has both A and B function (Figure 4). Thus class I1 duplications can form ac' triplications. However, in class I11 triplications, the center and (right segments cannot carry a functional B cistron since the duplication's left-hand end point is in that cistron. Hence class 111 duplications do not form ac' triplications. The properties for operational classification are summarized in Table 4. I n Table 5, the duplications analyzed by PARMA,INGRAHAM and SNYDER(1972) are classified.

Mapping experiments employing point mutations: The mapping experiments in this section are fundamentally crosses between a normal chromosome carrying a mutant allele (am) and a duplication carrying the wild-type allele ( a m + ) . Duplications recombinant for the interval defined by the breakpoint and the mutant site will be heterozygous if the site is included in the duplicated segment (i.e.. all recombinants will still have one copy of the wild-type allele). If 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 the right end point of class I I duplications (Method A): An ac' triplication is isolated, as described by PARMA,INGRAHAM and SNYDER (1972), from each duplication strain to be tested. From the acr triplications, r d P 8 ~ J 1 0 1and rdP8.rl589 duplications are segregated by asymmetrical pairing and recombination (Figure 5 ) . These latter duplications have the same end points as the original, are acriflavin resistant, but are unable to grow on a lysogen since neither type possesses both a functional A and B cistron. To localize the right-hand end point of the repeated segment, an rdP8 r1589 (or r d P 8 ~ J 1 0 1 duplication ) is crossed to an amber mutant situated to the right of the rII region (Figures 6a and 6b). Recombinant rdP8.r + duplications, which arise when the normal genome pairs

168

D. H. PARMA A N D M. SNYDER

TABLE 5 Classification of tandem duplications

Duplication

Fraction of progeny platingt

Mutation index on' CR/Xh f ac CR(Xh) f ac

+ ac

Class

x DplA DPGAS Dp8A DpQA Dp9D DpllC Dpl6A Dpl8A Dpl9A Dp2OA r48 amB262uc41

105 x 105 1.1 0.69 1.9 16 1.9 1.3 0.59 0.40 2.8 1.9 0.55 0.32 1.61 0.028 1.26 1.1 2.75 2.2 1.4 1.3 1.23 1.36 0.699 X 105 0.708 x

on CR(XL)

+ +

-rJlOl I

I

1

39

11 11 11

60'

I

60

++ +

1

B

b

I

A

I

I

60

I

1

I

I I

I I I 39

FIGURE13.-Alternative interpretations of end point mapping data. The duplication in 13a is drawn with its end point in the E4Q9-IC300 interval. However, the duplication in 13b, which has a deletion beginning in the E4Q9-E300 interval and eliminating all of gene 39, gives indentical mapping results. The latter alternative, while not rigorously excluded, is considered to be unlikely.

181

T A N D E M DUPLICATIONS

0

0

0

0

0

0

00

U 0

a0 m

0.10 Oh0 0130 2 x [JlOl SEGREGATION FREQUENCY

0140

%

io

$0 30 bo 2 x 1I589 SEGREGATION FREQUENCY

SO

FIGURE 14.-Segregation frequency as a function of duplication size. Segregation frequency is the fraction of r segregants/total progeny. Operationally, it is calculated as r segregants/ INGRAHAM and (plaque formers f r segregants) from the single cycle growth data of PARMA, SNYDER (1972, Table 1). The r segregants are included in the denominator twice (note, they are accounted for once in plaque formers) to account for inviable particles which occur with the same frequency as r segregants (PARMA,INGRAHAM and SNYDER 1972). For plotting, the r segregation frequency is doubled to account for triplications (inviable particles). In Figure I&, 2 x rJlOl segregation frequency is plotted versus the position of left end points; these segregants arise by recombination between rJl0l and the duplication’s left end. The origin is the left end of rJ101. In 14b, the plot is 2 x r1589 segregation frequency against the position of right end points; these segregants arise by recombination between ~ 1 5 8 9and the duplication’s right end. The origin is the right end of r1589. Intervals are arbitrarily drawn of equal size. D p l l C i s excluded from the figure since rJlOl maps in the left and A589 in the right half and therefore the segments cannot be properly aligned. The rJl0l segregation frequency is rJlOl segregants] (plaque formers f total r segregants). The r1589 segregation frequency is analogous. The data indicate a direct relationship between length and segregation frequency.

sidered not to be included in any of the duplications (Dp2OA is a possible exception). Figure 13 illustrates a possible source of error in interpreting our end point mapping data. Duplication strains are known to carry deletions which compensate for the otherwise increased length of a duplication genome (WEIL and TERZAGHI 1970; PARMA, INGRAHAM and SNYDER1972). In mapping the righthand end point of a duplication which carries a deletion in the left half between the rII deletion and the duplicated segment’s break point, the left end of the deletion may be mistaken for the duplication’s right-hand end point, if the deletion extends beyond our rII-most-distal markers. Although we consider this alternative structure to be intrinsically less likely, the two cannot be readily distinguished by genetic tests with available mutants but can be rigorously resolved by heteroduplex mapping of in vitro hybrid DNA molecules. In Figure 14, a plot of segregation frequency versus length shows a direct relationship. Extrapolation of this observation suggests that end points within

182

D. H. P A R M A A N D M. SNYDER

the same interval that have significantly different segregation frequencies are most probably at different sites. For example, the left ends of Dp8A and DpSA, both of which occur in the rPB296-r638 interval, are probably at different sites (Figure 12). The direct relationship between length and segregation frequency observed here contrasts with the inverse relationship reported by GREEN(1953a, b) for two tandem duplications, Bxr and Bxr40k, in D. melanogaster. The significance of this difference awaits further investigation. From a comparison of the segregation frequencies and recombination data, it is apparent that segregation occurs much more frequently than expected on the genetic size of a duplication as suggested by PARMA, INGRAHAM and SNYDER (1972). We are continuing our investigations of this phenomenon. Tandem duplications of defined genetic constitution off er a number of possibilities for studying gene action such as gene dosage effects, control of gene expression by removing a gene from its normal regulatory elements, position effects, and evolution. Dp8A presents a possible candidate for the latter, f o r in this duplication the left portion of gene 60 is spliced into the D region, denoted 60D. Gene 60D appears to have retained limited functional activity, as evidenced by discrepancies between functional and genetic end point mapping data (Table 6), although alternative explanations have not been excluded. By eliminating gene 60 from Dp%A,it may be feasible to study the changes involved in making 60D a more efficiently functioning gene. The order of mutant sites in the A cistron, gene 60 and gene 39 determined by duplication mapping is rHB84, rHB118, right end of rNB2226, (E429, E300, E1217, E594), E416 (E26, E566, E556, E1204, NG604), and E205, where the mutants within parentheses are not ordered with respect to one another. This order is in agreement with that obtained by conventional genetic mapping. This research was supported by NSF Grant No. GB-22611 and NIH Grant No. AI-09943. The senior author wishes to thank MINOUCHE PARMAfor her dogged loyalty and theoretical contributions to the experiments reported here. LITERATURE CITED

BAUTZ,F. A. and E. K. F. BAUTZ,1967 Mapping of deletions in a non-essential region of the phage T4 genome. J. Molec. Biol. 2 8 : 346-356. CHASE,M. and A. H. DOERMANN, 1958 High negative interference over short segments of the genetic structure of bacteriophage T4. Genetics 43 : 332-353. DOERMANN, A. H. and L. BOEHNER, 1970 The identification of complex genotypes in bacteriophage T4. I: Methods. Genetics 66:417-428.

A. H. and M. B. HILL,1953 Genetic structure of bacteriophage T4 as described by DOERMANN, recombination studies of factors influencing plaque morphology. Genetics 38: 79-90. DOVE,W. F., 1968 The extent of rII deletions in phage T4. Genet. Res., Camb. 11: 213-214. EPSTEIN,R. H., 1958 A study of multiplicity reactivation in bacteriophage T4. Virology 6: 382404. GREEN,M. M., 1953a The Beadex locus in Drosophila melanogasier: on the nature of the 195313 The Beadex locus in Drosophila mutants B z r and Bz'. Genetics 38: 91-105. -, melanogaster: genetic analysis of the mutant Bxr49k. Z. Verebungsl. 85: 4 3 5 4 .

TANDEM DUPLICATIONS

183

PARMA, D. H., I N 9 Acriflavin resistant rII deletions of bacteriophage T4. Genet. Res., Camb. 13: 329-331. PARMA, D. H. and L. J. INGRAHAM, 1970 Tandem duplications in bacteriophage T4D. Genetics (Suppl. 2) 64: 549-550. PARMA, D. H., L. J. INGRAHAM and M. SNYDER, 1972 Tandem duplications of the rII region of bacteriophage T4D. Genetics 71: 319-335. SYMONDS, N., P. VAN DEN ENDE,A. DURSTON and P. WHITE+ 1972 The structure of rII diploids of phage T4. Molec. Gen. Genet. 116 :223-238. WEIL,J., B. TERZAGHI and J. CRASEMANN, 1965 Partial diploidy in phage T4. Genetics 52: 683-693.

WEIL, J. and B. TERZAGHI, 1970 The correlated occurrence of duplications and deletions in phage T4. Virology 42: 234-237.