J. Mol. Biol. (1975) 91, 147-152

Tandem Genetic Duplications

in Phage Lambda

IV. The Locations of Spontaneously Arising Tandem Duplications SCOTT

W. EMMONS~ AND JOHN 0. THOMAS

Department of Biochemistr?J Stanford University .Me&cal School Stanford, C&&j. 94305, U.S.A. (Received 23 April 1974) Twenty-four spontaneously arising, long DNA addition derivatives of phage lambda have been isolated by two methods (one physical, one genetic) based on phage DNA content. All are shown to contain a tandem duplication of phage DNA by a number of criteria. The location of the duplicated segment in each has been determined by electron microscopy of DNA hereroduplexes. The duplications are found to lie at random throughout the chromosome, with no preferential locations for endpoints. This rules out the possibility that duplications are formed by crossing-over at regions of homology on the phage chromosome.

1. Introduction In the preceding paper by Emmons, MacCosham & Baldwin (1975), it is shown that tandem duplications are produced during the growth of bacteriophage lambda with surprisingly high frequency. This frequency was found to be unaffected by mutations in the bacterial and phage recombination genes, ruling out the possibility that tandem duplications are produced as a result of ordinary genetic recombination between short homologous regions of the phage chromosome. Earlier electron microscope mapping of a small number of tandem duplications in phage tde133 (Busse & Baldwin, 1972) suggested the existence of hot spots on the phage chromosome for the production of tandem duplications. Phage tde133 is a hybrid phage which derives the left half of its chromosome from phage h and its right half from phage $80. It makes a tiny plaque, and phage containing tandem duplications were isolated as large-plaque revertants. In the present work, we wished to study the production of tandem duplications in an all-X phage, so as to take advantage of t.he extensive h genetics. We also wished to isolate duplications by non-selective methods based on the DNA content of the phage particle. We have mapped a large collection of duplication phages obtained in this way and find a random distribution of duplication endpoints scattered throughout the chromosome.

2. Materials and Methods (a) Phge

and bacterial

Phage Ximm2 1, a hybrid phage containing Liedke-Kulke & Kaiser (1967) via R. Davis. are described in the preceding paper.

8tTaim

the immunity region of 421, is 21 hy 1 of Other phage and bacterial strains used here

t Present address: Carnegie Institution of Washington, versity Parkway, Baltimore, Md 21210, U.S.A. 147

Dept of Embryology,

115 West Uni-

148

S. W.

EMMONS

AND

J. 0.

THOMAS

(b) Media The media

used are described

in the preceding

(c) Isolation

paper.

of tandem dupltiiwna

(i) From CsCl density gradients Density purified XcI26b221red270 was grown through a single lytic cycle on W3lOlrecA 13 and the progeny phage banded on a C&l density gradient. These procedures are described in detail in the preceding paper. Single-plaque isolates on W3lOlrrecA were made from the region of the gradient expected to contain addition mutants with DNA additions greater than 10% the length of h. A five-plate stock of each isolate was prepared on strain C600, the phage pelleted, and the resuspended pellets banded overnight in CsCl of mean density 1.5 g/cm3. Centrifugation was at 35,000 revs/min in a Type 40 fixed-angle rotor. Mutant phages carrying DNA additions were readily identified by the position at which they banded in this gradient. Sixty isolates were examined in this way and 14 proved to carry DNA additions. Most of these additions were further characterizable as tandem duplications by the presence of multiple phage bands: duplication mutants segregate both the parental phage and triplications, etc. by unequal crossing-over on growth in a recomet al., 1971). On further examination by bination-proficient host such as C600 (Bellett electron microscopy of DNA heteroduplexes, all proved to be tandem duplications. Phage stocks obtained from these gradients by removing the phage bands from the side of the tube with a syringe were used for density determination and heteroduplex mapping as described below. Two duplications (a66 and a67) were isolated during the analysis of density purified stocks of AcI26b221red270 (see preceding paper, Fig. l(b)). The nomenclature used here to denote tandem duplications (a followed by an isolation number) is that suggested by Emmons (1974). (ii) Genetic selection for tandem duplications In the preceding paper we describe a mutant of Eecherichiu coli (GLl) which does not support the growth of h deletion phages. The exclusion is baaed non-specifically on the DNA content of the phage particle (unpublished data) and the strain may be used to select directly for tandem duplication derivatives of deletion phages. This has been done in two ways here. In the i%st method, a lysate of XcI26b221red270 which has not been fractionated by density is plated directly on GLl. Plaques are picked and analyzed as described in the previous section. In this way, tendem duplications and point mutations of the deletion parent which plate on GLl are recovered with approximately equal frequencies. Three duplications described here (a106-12, a106-16, and a106-19) were isolated in this way. In the second method, hcI26b221red270, purified by density, is plated on a mixed indicator containing W3101 and GLl in the ratio 10: 1. Phage are allowed to adsorb to W3101, and GLl is added just before plating. On this indicator plaques of XoI26b221~ed270 are turbid; however, between 1% and 10% of the plaques contain clear spots or sectors. These are picked, the phage replated on the mixed indicator, and clear plaques selected. Five out of five clear plaques of independent origin selected in this way proved to be tandem duplications (a105-1 to ~105-5). (d) Determination

of length of tandem

duplications

The length of the duplicated region in tandem duplication phages was calculated aa the difference between the DNA lengths of the duplication phage and the parental hb221. The length of r\b221 is taken to be 0.777 X lengths (Davis & Davidson, 1968). DNA lengths were calculated from phage buoyant densities ae described previously (Bellett et al., 1971; Emmons, 1974).

(e) Electron microscopy

of DNA

heteroduplexes

DNA heteroduplexes were prepared and mounted for electron microscopy using the formamide technique of Davis et al. (197 1). Stained and shadowed grids were examined and photographed with a Philips EM300 electron microscope. Photographs were measured

LOCATION

149

OF DUPLICATIONS

by projeuting them onto the surface of a Hewlett Packard 9864A Digitizer and calculating oontour lengths with a Hewlett Packard 9810A Calculator. Lengths (in units of h+ length) were oalmlated relative to the average length of several relaxed circles of phage PM2 DNA appearing in the same photograph with the DNA heteroduplexes. The ratio of the length of X DNA to the length of PM2 DNA was measured to be 4303f0.03. The error indioatod in mesaured quantities is the standard error of the mean, o/N *, where (I is the standard deviation of the measurements and N the number of measurements. Precise duplication endpoints are the result of measuring between 10 and 50 heteroduplexes. (f) Procedure. for mapping tandem duplicd&one Tandem duplications give rise to movable loops in heteroduplexes with unduplicated DNA (Busse & Baldwin, 1972). Such heteroduplexes can be used to determine roughly the position on the chromosome of the duplicated segment but not the exact location of its endpoints. Duplication endpoints may be accurately determined from heteroduplexes constructed from two different tandem duplications chosen so that some sequences are duplicated by both (Busse & Baldwin, 1972). These heteroduplexes contain fixed loops or bubbles which determine the position of the novel joint (Hershey, 1970) as illustrated in Figure 1. The position of the novel joint relative to one end of the chromosome and the ength of the duplication (determined from phage densities 8s described above) determine he endpoints of the duplicated segment. o

bcdef

Parental sequence

.LR(I)

a b e d,b

c

d,e

f

Duplication I

+----II-LL(2)

--4

o bcdecdef Duplication 2 kf(2)-1

‘Z

J(2)

’

a b c d, b cdef Heteroduplex -A

abcdUcdef -Iei------B-’

mapping of overlapping tandem duplioetions. The lengths of the doubleFro. 1. Heteroduplex stranded regious of the heteroduplex, designated A and B, ere measured 8s fractions of the 1engt.h of A using double-stranded mole&es of phage PM2 DNA in the same photograph 8s a Iength stand8rd. Then the left tlnd right&and endpoints of the two duplications are given by the following relations: L,(l) = A - Z(l), L,(l) = A, LL(2) = 1 -B, L,(2) = 1 - B + Z(2); 1(l) is the length of the duplicated segment in duplication 1, determined from the phage buoyant density. J( 1) is the position of the novel joint in duplication 1.

The general scheme for mapping a large number of duplications was as follows: one of the duplications was chosen at random and the heteroduplex with Gmmtl was examined. The bubble resulting from the immtl substitution identified the right end of the heteroduplex, and the location of the duplicated region was determined from the position of the movable duplication loop. This duplication was then used to map subsequent duplications. In these heteroduplexes, the loop of the first duplioation served to orientate the heteroduplex and the position of the seoond was roughly determined. If the two duplioations happened to overlap, then auourate endpoints were immediately obtained. After rough positions of all the duplications were obtained, heteroduplexes between overlapping pairs were examined, mined. 11

until

the endpoints

of all of the duplications

had been accurately

deter-

S. W.

150

EMMONS

AND

J. 0.

THOMAS

3. Results and Discussion Twenty-four Iong DNA addition mutants of hcI266221red270 have been isolated and studied in detail. All contain tandem duplications of phage DNA by the following criteria : they segregate multiple, evenly-spaced phage bands in CsCl density gradients when grown under recombination-proficient conditions (Bellett et al., 1971) ; they give rise to a movable loop in a heteroduplex with parental DNA; they give rise to fixed loops or bubbles in heteroduplexes with overlapping duplications (Busse & Baldwin, 1972). In all cases the duplicated segments are both oriented in the same direction and there is only a single novel joint. In heteroduplexes between completely overlapping duplications even a small amount (>50 base pairs) of unduplicated phage or foreign DNA at the novel joint could have been detected but was not. TABLE

1

Length.8 and endpoints of tandem duplications Duplioation phrtge number

LS

LRS

al6

0.183

a3bb a38 a40 a41 a42 a46 a47 a50

0.159 0.100 0.170 0.106 0.173 0.167 0.158 0.132

a51 a54 a55 a60

a62

0.151 0.128 0.147 0.127 0.167

a66 a67 alOba105-2 a105-3 a105-4

0.065 0.062 0.159 0.100 0.170 0.144

(not accurately determined) 0.264 0.656 0.949 0.049 0.298 0.701 0.277 0.383 0.823 0.996 0.815 0.972 0.842 1.00 (not eacurcttely determined) 0.202 0.353 0.866 0.994 0.747 0.894 0.859 0.986 (not fwxurately determined) 0.233 O-298 0.173 0.235 0.028 0.187 0.806 0.906 0,829 0.999 0.191 0.335

alOb-

0.146

0.131

0.276

a106-12

0.096

0.186

0.282

a106-15 a106-19

0.229

0,314 0.021

0.766 0.202

0.181

Heteroduplexes examined a16/a106-19 a3bb/a106-12 a38/alO6-19 a40/alOb-4 adl/abl a42jabb a46/abb a47/abb, a47/alOb-3 t abl/al06-15, ab4/a42 abb/a47 a60/a47 §

abl/a41

a66/a67 a67/a66 a105l/alOb-4 alO&2/alOb-3 alOb-3/a47, alOb-3/a106-2 alOb-4/a40, a105-41 alOb-1, alOb-4/a106-6 alOb-6/alOb-4 alOb-b/ a106-19 a106-12/a356, alOg-121 a106-4 alO&lS/abl alOB-19/a38, a106-19/ alOb-5

t 2 is the length of the duplicated region; oalculeted from phage buoyant densities as described in Materials and Methods. $ LL and La are, respectively, the left-hand endpoint and the right-hand endpoine of the duplioation (see Fig. 1). 3 These duplications give rise to single-stranded tails on the right. end of hekeroduplexes, indioating t,hey map very close to the right end of the chromosome.

LOCATION

A W BCDEFZUVGT

HH /C--i

HMLK

HYHHHHHHHIHH a 3%

I

Hti

a35bbp a4OV a41+

151

OF DUPLICATIONS

43

J

H f----4 i

I

a5le a66H a67H

“O&d

N mu cl cn OP

-IH

+g

i- i i- + A6,------o55tI

QSR

a38t

Y

054H

a42*-a46l-A 047t-------a50 a62 060-i

I

0105-2H alO5-3V

alOg-I+---+ 01054t-------i al05-5m alO6-12al06-15*

-7i

olO6-19+-----------I

d FIQ. 2. The location

of 24 tandem

i duplications

in phage lambda.

The endpoints of the duplicated region in these 24 isolates are given in Table 1 and Figure 2. The endpoints of ail except a16, a50, and a62 are determined to within 0.01 I\ lengths. Duplication phage al6 grows very poorly and was not obtained in sufficient quantities to determine precise endpoints. Duplications ~42, a47, a50 and a62 give rise either to movable loops or singlestranded tails on the right-hand end of heteroduplexes. Apparently their right-hand endpoint lies so close to the right end of the chromosome that the movable loop can migrate off the end of the heteroduplex. Duplication a38 duplicates the site on the chromosome where the cohesive ends are formed. This phage has interesting properties which have been described (Emmons, 1974). In heteroduplexes with parental DNA, a38 gives rise to single-stranded tails which are predominantly on the left hand of the chromosome and occasionally on the right end. This property distinguishes a38 from a42, ~47, a50 and a62, which give rise to single-stranded tails only on the right end of the chromosome. The striking feature of Figure 2 is the apparent randomness of the distribution of the duplications throughout the chromosome. There is no region of the chromosome which is not covered by at least one of the duplications. The slight excess of duplications covering the QSR region is accounted for by the fact that several of these duplications form large plaques, and large plaques were selectively chosen during the isolation of the duplications. The endpoints of the duplicated regions are scattered across the chromosome, and the possibility that an endpoint is held in common by more than one duplication exists in only a few cases. The large number of endpoints implies that very little, if any, sequence homology is necessary to form the novel joint.

162

S. W. EMMONS

AND

J. 0. THOMAS

The question of sequence specificity at the novel joint remains open, however, for phage tdel33. As we have shown in the preceding paper, duplication production is stimulated by one of the gene products of tdel33, and this could be a site-specific pathway. The authors thank Dr R. L. Baldwin for the guidance he has provided throughout the course of this work, and Drs N. C. Franklin and A. D. Kaiser for their comments on the manuscript. This work was supported by research grants from the U.S. National Institutes of Health (grant GMAM19,983-13) and National Science Foundation (grant GB-35432X) to R. L. Baldwin. One author (S. E.) is a U.S. Public Health Service predoctoral trainee; the other author (J. T.) is supported by a Damon Runyan cancer research fellowship. REFERENCES Bellett, A. J. D., Busse, H. G. & Baldwin, R. L. (1971). In The Bactmiophuge Lambda (Hershey, A. D., ed.), pp. 601-513, Cold Spring Harbor Press, New York. Busse, H. G. & Baldwin, R. L. (1972). J. Mol. Biol. 65, 401-412. Davis, R. W. & Davidson, N. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 243-250. Davis, R. W., Simon, M. & Davidson, N. (1971). In Methods in Enzymology, VoZ. XXI, Nucleic Acids, Part D (Grossman, L., ed.), p. 413, Academic Press, New York and London. Emmons, S. W. (1974). J. Mol. BioZ. 83, 511-525. Emmons, S. W., MacCosham, V. & Baldwin, R. L. (1975). J. Mol. B&Z. 91, 133-146. Hershey, A. D. (1970). Yearb. Camzeg& In&n., 69,117-722. Liedke-Kulke, M. & Kaiser, A. D. (1967). Virology, 32, 476-481.