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Sequential replication of the Bacillus subtilis chromosome
J. Mol. Biol. (1967) 27, 349-368
Sequential Replication
of the Bacillus subtilis Chromosome
IV.7 Genetic Mapping by Density Transfer Experiment AIDEEN
O’SULLIVAN
AND NOBORU
SUEOKA
Department of Biology, Princeton University Princeton, New Jersey, U.S.A. (Received 13 December 1966, and in revised form
3 March 1967)
Genetic markers near the replication origin of the chromosome have been mapped in two strains of Bacillus sub&s, W23 and W 168, which differ in replication control. A synchronization of chromosome replication during spore germination was used. Two types of density transfer experiments of spores were performed: one, the germination of deuterium oxide-grown spores in a rich aqueous medium and the other, the germination of spores of a thymine-requiring strain in a medium containing 5-bromouracil. Samples were taken at various times during germination and their lysates were centrifuged in a density gradient. The transfer of genetic markers from non-replicated, half-replicated and fully replicated DNA was followed by transformation assays. A theory of mapping applicable to replication synchronization of the chromosome and density transfer experiments (synchro-transfer analysis) has been developed and applied to the present data with special emphasis on markers close to the replication origin. This method of mapping has been extended to the whole chromosome, and the relative distances between markers was estimated by statistical analysis of the transformation data. There was no difference in the replication origin and in the marker order
between the two strains.
1. Introduction The replication order of markers in the Bacillus subtilis W23 chromosome has been determined by marker frequency analysis in transformation (Yoshikawa & Sueoka, 1963a; Sueoka & Yoshikawa, 1965) and confirmed (Yoshikawa C%Sueoka, 19633) by following the order of transfer of genetic markers from heavy to hybrid density positions when synchronized heavy cells (stationary phase in Da0 medium) of B. subtilis W23 are diluted into a light medium. For reasons discussed in these papers, the most likely representation of the replication order is a linear genetic map, which shows that the chromosome replicates sequentially from the replication origin to the terminus, Using strain W168, the replication order was found to be identical to that of strain W23 when spore DNA of st,rain W168 was taken to represent that of completed chromosomes. However, it was found that in strain W168 the marker ratio did not correspond to that expected for completed chromosomes, implying that stationary cells did not contain completed chromosomes and indicating a difference in control of replication (Yoshikawa, O’Sldlivan & Sueoka, 1964). t fart
III
of this series is Yoshikawa,
O’Sullivan 349
& Sueoka, 1964.
360
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This report presents a more extensive study of genetic markers near the origin of replication in both strains, and describes a method of chromosome mapping by transfer experiments. Synchronization of chromosome replication during sport: & Sueoka, 1964) germination (Wake, 1963; Yoshikawa et al., 1964; Oishi, Yoshikawa was used. The results present a confirmation and extension of the B. subtilis map, with particular emphasis on markers near the origin of replication.
2. Materials and Methods (a) Strains
Donor DNA for transformation was prepared and 23th~~his (from F. Rothman). Recipient strains for transformation: 168ade33 (Mu33) 108ade34 (Mu34) 108Eeu-adel2 (Mu8u12) 168leu-met-ade6
(MuSLl5u6)
1681e~-met-&e16
(Mu8u5u16)
168leu-met-ode11 168auc-i&met
(from
Ural(L)-hial(L)&y2(L) met3(L)&y2(L) PWL) 168Zeu-met-thr 168lewmet-hia 168&a 168lewmet-ileu 1681ys-phe In order to avoid confusion, denoted as (L). Other mutants
from the following
strains: W168, W23
(MuSu5u17)
Spizizen, met(u5) marker formation by Dr M. Masters) (SB5 from Lederberg) (SB26 from Lederberg) (SB 133 from Lederberg) (MuSu5u5) (Mu8u5u2) (Mu36) (Mu8u5ul) (Mu12u17) mutants isolated in the laboratory were isolated in our laboratory.
introduced
by
of J. Lederberg
trans-
are
(b) Media Medium C (Spizizen minimal salts medium) contains, per liter: 14.0 g KzHPO,; 6.0 g KH,PO,; 2.0 g (NH,),SOI; 1 g trisodium citrate,2H,O; 0.2 g MgSO,,7H,O; 5 g glucose. Medium C+ . Medium C supplemented with 500 mg Casamino acids (Difco Labs); 60 mg L-tryptophan; 60 mg base requirement; 100 mg ammo acid requirement per liter. Medium C++. Medium C supplemented with 100 mg Casamino acids (Difco); 5 mg n-tryptophan; 5 mg L-histidine; 10 mg base requirement per liter; no extra amino acid is added for mutant requirement. Low-phosphate-D20-32P-kxbeled sporulation plates. 22 mg MgSO,; 450 mg (NH&SO,; 1.13 g potato extract (Difco); 10 mg CaCl,; 315 mg KsHPO,; 135 mg KHzPO,; 225 mg (pH 7.0) (all dissolved in DzO and sodium citrate; 8 mc aaP; 1.13 g glucose; 0.05 M-T& dried in vucuo), DzO (99.7%) 225 ml. Penassay germination medium. Antibiotic medium 3 for penicillin assay (Difco), 11.7 g per liter. [3H]Thymine spwdation plates. 3H-labeled spores of 23thy-his were prepared on plates of the following composition: 400 ml. medium C; 40 mg L-histidine; 2 mg thymine; 2 g potato extract (Difco); 16 mg MgCI,; 1.6 mg CaCl,; 16 g agar. After autoclaving, sterile glucose (0.5%) and [3H]thymine (1 mc, specific activity 6.7 c/m-mole) were added. Bromouracil germination medium. Medium C; 2.5 pg thymine; 100 pg n-histidine; 100 pg L-alanine; 500 pg C&amino acids (Difco); 200 pg yeast extract (Difco); 25 pg B-bromouracil (Calbiochem.); 5 mg glucose/ml. DzO sporukztion pkztes. The following were suspended in 10 ml. of D,O, dried in vacua, redissolved in 1 liter distilled DsO, sealed, autoclaved and poured into plastic plates: 14.0 g
SYNCHRONOUS
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351
1.0 g sodium citrate,2HzO; 4 mg M&I,; 6.0 g potato extract (Difco); 50 g glucose, and 40 g/l. agar. Rich low-phosphate 32P-labeled germination medium. 8 g Nutrient broth (Difco); 1.5 g Bacto yeast extract (Difco); 3.5 g NaCl; 1 M-Tris buffer (pH 7~3)~ 50 ml. per 1. 9.8 mc of 3aP (neutralized He3zP0,) was added in 30 ml. of the medium.
6g KzHPO,; 97.6 g MgSO,;
KH,PO,; 40 mg C&l,;
Exp. 1. Replication
2.0 g (NH&SO,;
(c) Method.9 order of adenine markers in atrain W168, by DsO transfer experiment spores were prepared by pre-adapting B. subtilis W168 to growth in
3aP-D,0-labeled D,O medium by serial transfer through 40, 65, 95 and 100% D,O media. An overnight culture of cells was grown in D,O-C+ medium and plated (0.2 n&/plate) on D,0-3aPlabeled sporulation plates. Spores were prepared after 10 days incubation at 37°C. The spores were scraped off the plates, filtered through Whatman no. 1 filter paper, washed twice with sterile distilled water, purified by incubation with egg white lysozyme (Worthington, 1 mg/ml., 50 mm, 37”C), and subsequently with sodium lauryl sulfate (2%, 30 min, 37”C), and finally washed several times with sterile distilled water. The spores were germinated in H,O-Penassay germination medium, cell samples were taken at 33, 43 and 53 min after addition of spores, samples were immediately hilled by heat (60°C, 10 mm), cooled in ice-water, lysed with lysozyme (1 mg/ml., 45 min, 37°C) and sodium lauryl sulfate (2%, 30 min, 37’C). The lysates were centrifuged in a C&l densitygradient (final density 1.70 g/cm3) in Tris (0.01 M)-EDTA (0.001 M) buffer (pH 8.4) for 3 days at 25”C, 35,000 rev./min in SW39 rotor of a Spinco model L ultracentrifuge. Drops were collected from the bottom of the tube, each drop was diluted with 1 ml. sterile SSC (O-15 M-NaCl, 0.015 M-Sodium citrate, pH 7.0). The 32P profile was obtained by removing 0.2 ml. from each tube and assaying for 32P activity by precipitation-in the presence of carrier DNA (200 pg crude salmon sperm DNA/ml.)-with 10% trichloroacetic acid. The precipitate was washed on a membrane filter (coarse, Schleicher & Schuell, Keene, N.H.), dried, and radioactivity counted in a liquid-scintillation counter. Transforming activity was determined by accurately removing 0.1 ml. (or 0.05 ml.) from each tube, adding 1 ml. (or 0.5 ml.) of competent recipient cells and plating on selective media after 40 mm incubation and the addition of DNase. Competent cells were prepared (Anagnostopoulos & Spizizen, 1961) by growing cells in C+ medium until the end of the logarithmic growth phase, diluting l/10 into C++ medium and incubating at 37°C for 90 min. Competent cells were either freshly prepared or stored at tenfold concentration in 50/ glucose at -80°C.
Exp. 2. Replication experiment
order
of
adenine
markers
in
strain
23 by bromouracil
transfer
3H-labeled spores of 23thy-his were prepared by 4 days incubation at 37°C on [3H]thymine-sporulation plates. The spores were purified as described for 3aP-Dz0 spores. The 3H-labeled spores were germinated in bromouracil germination medium. Samples were removed at 45, 52, 55, 70 and 85 mm. Subsequent steps were similar to those described for exp. 1.
Exp. 3. MappirLg of B. subtilis
chromosome
by DzO transfer experiment
D,O-treated spores of WI68 were prepared on D,O sporulation plates. On washing, the crude spores formed three layers. Each fraction was purified separately and tested for germination; the fraction giving the fastest germination curve was used for the experiment. The spores were germinated in rich, low-phosphate medium containing 3zP. 6-ml. samples were taken at 50, 60, 73, 85, 100 and 115 min after addition of spores. Subsequent steps were similar to those described for exp. 1.
3. Results Since there is a difference in the control of chromosome replication between B. subt&s strains W23 and W168, it was of interest to determine whether or not markers near the origin of replication were similar. It has been known that a purine-requiring
352
A.
O’SULLIVAN
AND
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SUEOKA
mutant (ade6) occurs within 10% of the replication origin in bot,h strains (Yoshikawa & Sueoka, 1963a). When the density of B. su.bti& DNA was exa.m.ined in a preparotivc: CsCl density-gradient, it was observed that various purinr: markers fill into tfwo density groups (henceforth referred to as groups 1 and 2): indicating that there arc at least two purine regions on the chromosome (O’Sullivan, Yoshikawa & Sucoka., unpublished data). The replication order of various adenine (actually purine) markers has been shown by a D,O-H,O transfer experiment in which D,0-32P-labeled spores of strain W168 were germinated in a light Penassay medium. Samples were taken very shortly after the commencement of DNA replication-previously estimated by [3H]thymine incorporation. (In the germinating spore system it is not necessary to use a thyminerequiring mutant for thymine incorporation.) The replication order of markers was determined by centrifuging the lysed samples in C&I, density 1.70 g/cm3, and testing the heavy (parental) and hybrid (newly replicated) DNA regions for transforming activity for the different markers at increasing times after the start of the DNA replication. The sample in Pig. 1 shows that in strain W168 the markers ade16 and ade17 (group 1 density) are about two-thirds replicated at 53 minutes, whereas the markers ade6 (group 2) and met are still completely in the heavy-density peak. In this experiment, the 32P count was not sufficient to estimate the percentage of DNA which had replicated at this time.
Drop no. FIG. 1. Replication
of various
adenine
markers
in D20
transfer
experiment.
D-32P-labeled spores of WI68 were germinated in light Penassay medium. This sample was does not allow us to estimate taken 53 min after start of germination. The low a2P radioactivity 69 and 70) is not significant’ly the replicated amount of DNA. A small aaP peak (drop numbers larger than the background noise. saP radioactivity (--e--e--). adelG (-x-x-) and utZe17 (group 2); Distribution of markers : mZe6 (-o-o-) (-cl--o-) (group 1); met (-O-O-).
SYNCHRONOUS
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353
OF CHROMOSOME
Figures 2 and 3 show that in strain W23 a similar replication order is obtained when [3H]thymine-labeled spores of 23thy-his are germinated in a medium containing 5-bromouracil. In these experiments, parental DNA is in the light-density band and newly replicated DNA in the hybrid region. Figure 2 shows that the markers adel6 and a&l7 (group 1) are transferred, whereas ade6 and a&34 (group 2) are not. In
Parental
Strain 23
,ade34 -32
3000-
2500-
.E E 9u
zoooade6
,I ISOO-
IOOO-
500 -
0 50
52
FIG. 2. [3H]Thymine-labeled min. 3H radioactivity (-++--). adelS(-a-u-)andadel’l (group 2).
56
60 64 Drop no.
spores of 23th~~ks
(-A-A--)
germinated
6%
72
in 5.bromouracil
0 76
medium
for 50
(group l);ade6(-x-x-)andade34(-O-O-)
this sample, the number of ade16 transformants in the hybrid region is 40% of the total. The proportion of DNA which has replicated can be estimated from the distribution of 3H as approximately 3%. A shoulder seen in both the 3H profile and marker profile of ade6 and ade34 (Figs 2 and 3) deserves comment. This shoulder has been observed in nearly all 5-bromouracil transfer experiments. Moreover, all genetic markers can be found in the shoulder to an approximately equal extent, without
354
A. O’SULLIVAN
AND
N.
SUEOKA
regard to the position of markers on the chromosome. Together with the fact that the density of the shoulder is between the parental and hybrid peaks, the shoulder seems to represent DNA molecules which have undergone repair processes. A similar obscrvation was made by Yoshikawa (1965). In the present calculation of the extent of replication, therefore, shoulder activities have not been included.
r-----l,, Stmin23
Parenta I
6000 -
0
65
70
75.. Drop
FIa. 3. [eH]Thymino-l&&d
spores
of
80
BS
90
no.
23th~~hie germinated
in 5-bromowacil
medium
for
86 min. 3H radioactivity ade6 (-a--@-), fhf (-O-O--).
(--O--O--). c&e34 (-x-x-),
adel2 (-A-A---),
c&e33 (-B-m-)
(all
group
2);
Although synchrony in D,O transfer experiments is considerably better than that in 5-bromouracil, the density difference obtained is not sufficient to separate completely the 3H profiles of heavy and hybrid DNA. Figures 2 and 3 and Table 1 confirm the replication order of markers as being adel6, adel7 (group I), ade6, ade34 (group 2) with adel2, ode33 (group 31) and thr still unreplicated. The results of these experiments show that there are at least two groups of ade mutants, that ode markers in density group 1 are replicated earlier than those in group 2, that both groups are replicated before the thr marker, that the earliest known marker in B. sub&s is adel6, and that this marker is located within 2 or 3% of the origin of replication.
SYNCHRONOUS
REPLICATION TABLE
OF
355
CHROMOSOME
1
Marker distribution in the D,O --f H,O transfer experiment
DNA donor W168
Marker
o/o of total trrtnsformants in?
Total no. of transformants HH(oo)
HL(m)
LL(nn)
Sample 1, 50 min after germination
adel6 8uc(Spizizen) hisI adef3 Ural(L)
366 2459 1846 304 1695
21.3 33.4 40.3 62.7 97.9
73.5 66.6 59.7 37.3 2.1
5.2 0 0 0 0
Sample 2, 60 min after
adelf3 auc(Spizizen) ade6 thr metS(L)
133 1109 1360 97 258
10.1 15.7 22.4 31.2 70.1
752 71.8 67.2 66.7 29.9
14.7 12.5 10.4 2.1 0
Sample 4, 85 min after germination
adel6 suc(Spizizen) hial ade6 thr met3(L) leu PheW Ural(L) w/W)
81’7 2587 1913 102 1071 478 375 4051 2537 2588 832
6.3 12.4 8.4 15.2 6.9 12.6 11.8 11.8 17.8 28.1 33.9
35.1 37.6 44.1 65.4 65.7 65.7 74.3 77.6 81.5 69.7 63.2
58.6 50.0 47.4 29.4 27.5 21.8 13.9 10.6 0.7 2.1 2.9
449 13914 3328 14673 6366 2114 5671 4214 52270 6788
6.5 6.8 12.4 10.7 19.8 11.0 15.9 12.8 17.1 14.2
25.4 40.1 45.0 60.7 43.8 58.4 58.8 63.4 59.2 67.3
68.1 53.1 42.6 38.6 36.4 30.6 25.3 23.7 23.7 18.5
met
Sample 5, 100 min after qermination
de16
his met3 leu
Ural(L) lY8 ilva ww
7ne.t
ileu
t HH refers to the heavy density DNA region, HL to the hybrid density DNA region and LL to the light density DNA region. A more general way of designating parental, hybrid and fully replicated DNA ia shown in parentheses to eliminate the confusion of using different density transfer systems (e.g. D,O+H,O, H,O+D,O, bromouracil+T, T-tbromouracil). 00, Both strands are old (parental). on, One strand is old and the other new (hybrid). nn, Both strands are new (fully replicated).
Since chromosome synchrony was obtained during the germination of D,O-treated spores, this method was used to extend the map of the B. subtilis chromosome. In our germination conditions, re-initiation of daughter replication origins occurs before the first replication point reaches the terminus (dichotomous replication) (Yoshikawa et al., 1964; Oishi et al., 1964). Therefore, transfer of markers both from heavy to hybrid density and from hybrid to light density can be examined. D,O-treated 24
356
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O’SULLIVAN
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SUEOKA
spores of W168 were germinated in a light, rich, low-phosphate medium containing 32P and samples were taken throughout the course of one generation. The lysed samples were centrifuged with CsCl, the 32P count being used to locate the DNA region. Some examples of the results are shown in Figs 4 to 8. Figure 4 shows the replication order of early markers to be udel6, sue, &l(L) and ade6; Fig. 5 shows the replication order of a&6, thr, met3(L), leu; Fig. 6 shows the later markers leu, Ural(L), phe(L), tryZ(L) and met; Fig. 7 shows the replication order of his, metS(L), leu; Fig. 8 shows that ileu, ilva, met and tryB(L) are close together and that these
Hybrid
Ii
Strain 168
Drop no. Fm. 4. D,O transfer experiment. Replication order of markers is shown to be ode16 (-a-@-), (-u-m-) and ade6 (-O-O--). See also Table 1. (a) Transfer sample 1, 50 min after start of germination. (b) Transfer sample 4, 86 min after start of germination.
8~: (-x-x-),
h&l(L)
SYNCHRONOUS
20
1
I 1 Parenta I
,
,
REPLICATION
1
1
I
OF CHROMOSOME
&.$
18-
’
i
’
’
’
‘Stra’in
16b
Fully replicated
Drop
no.
FIG.6
Replication (-B--B-) germination.
order of markers and Zeu (-A--A-).
FIG. 5. Da0 transfer experiment. is shown to be ade6 (-@-a-), thr (-x-x-), m&3(L) T ransfer sample 4 (same aa Fig. 4(b)), 85 min after start of
FIQ. 6. D20 transfer experiment. Replication order is shown to be Zeu (-A-A-), urul(L) (-x-x-), phe(L) (-O--O-), tryl(L) (-O-O-) and met (-W-m-). Transfer sample 4 (same as Figs 4(b) and 5), 85 min after start of germination. See also Table 1.
358
A. O’SULLIVAN
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SUEOKA
18 16
t
G 5 %
-
Strain 168
22-
$
Fully replicated
18-
Parental
l4-
6’-
0
50
55
60
65
Drop no. FIG.8 FIG. 7. D,O transfer experiment. Replication order of markers is his (-O-O-), w&S(L) (-m-w--) Transfer sample 6, 100 min after start of germination. See also Table 1.
and Zeu (-A-A-).
FIG. 8. D20 transfer experiment. Replication order is shown to be urul(L) (-O-O--), ilwa (-x-x--), met (-•-•.r), try!Z(L) (-•--a---) and ileu (-A-A-). Transfer sample 5 (same as Fig. 7), lOOmin start of germination. See also Table 1.
after
SYNCHRONOUS
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359
CHROMOSOME
markers oannot be given their map positions from the results. This is not unexpected, since these are very late markers and synchrony deteriorates toward the end of one generation. The position of the auc marker (0.02) mapped by the present method agrees well with the results (0.013) of Masters BEPardee (1965) by marker-frequency analysis. The results of this experiment are summarized in Table 1, which shows the proportion of each marker in heavy, hybrid and light bands. The replication order, and hence presumably the map order, can be seen to be adel6, SW, hial( adeb, thr, his, met3(L), Zeu,urul(L), phe(L), [ilua, tryS(L), Zya,met, ileu]. The square brackets around the last group of markers indicate that their order is uncertain. In addition to the replication order, the relative distance between markers can be estimated from a statistical analysis of these results (using any one sample) by measuring either the distribution of a marker between heavy and hybrid regions or between hybrid and light regions, and relating it to the distribution of either two known markers or one known marker and the DNA (e.g. from the 32P count). These analyses are shown in Tables 2 to 5. (The method of calculation of the map position X will be found in the Appendix.) TABLE 2 Transfer sample 1 (see Table 1): HH --) HL andyais~ oh of transformsnts involved in heavy to hybrid trensfer which 8re in heavy (HH) and hybrid (HL)
Marker
adel6 auc(Spi2.) hisl(L) ode6
JWXoo)
HWGn)
22.5 33.4 40.3 62.7
77.5 66.6 59.7 37.3
Map position
(X)
O$ (reference marker) 0.02 0.04 0.07-i (reference marker)
t Used when X,, N 0 8nd X,, > 0. For symbols X,, and X0,, see Appendix. $ Adel6 is the closest marker to the replication origin so far obtained. Map position 0 is tern. porarily assigned to this marker. The position of ade6 is the average estim8te m8de by markerfrequency 8nalysis (H. Yoshikawa, M. N. Hayeshi & N. Sueoka, menuscript in preparation).
TABLE
3
Transfer sample 2 (see Table 1): HL + LL analyaes~ Marker
o/0 of transformants in HL end LL regions HWGJ
adel6 suc(Spiz.) de6 thr
83.6 85.2 86.6 97.0
t Not accurate due to the existence of three However, there is residual ctctivity of ads16 from adel6 velues in 18ter samples) which makes the symbols X0,, end X,,. see Appendix. $ An average estimate by merker-frequency Sueoka, mtwiusoript in preparation).
Map position
X
W-L”) 16.4 14.8 13.4 3.0
0 (reference marker) 0.02 0.06 0.271 (reference marker)
pe8ks of tr8nsforming activity (see Table 1). non-repliceting chromosomes of about 7% (see analysis of HL+LL somewhat justifiable. For analysis
(H. Yoshikawa,
M. N. Hayashi
& N.
360
A. O’SULLIVAN
AND
N.
SUEOKA
TABLE 4 Transfer
sample 4 (see Table 1): HH -+ HL and HL + LLT analyses o/0 of transformants
Marker
=Woo)
adel6 suc(Spiz.) on
HWL)
13.6
86.4
17.9 28.7 34.9
82.1 71.3 65.1
in HL and LL regions
HH and HL regions
HWLJ
Map position
(X)
w&n)
34.7 29.5 24.9 16.4 12.0
65.3 70-5 75.1 83.6 88.0
0.11 0.27$ (reference) 0.40 0.623 (reference) 0.78 0.73 0.91 1.003 (reference)
t Used when X,, N 0 and X,. > X,,. The results are not accurate because of the large values of (I. $ Average estimates by marker-frequency analysis (H. Yoshikawa, M. N. Hayashi & N. Sueoka, manuscript in preparation).
TABLED Transfer
Marker
sample 5 (see Table 1): HL + LL analysis o/0 of transformants in HL and LL regions HWLJ
met3(L) leu lY8 ilVlZ
met trYw) ileu
51.4 56.8 65.6 69.9 71.4 72.8 78.4
t Average estimates by marker-frequency manuscript in preparation).
Map position
(X)
w-&J
48.6 43,2 34.4 30.1 28.6 27.2 21.6 analysis
0.26 0.62t (reference) 0.92 0.97 1.007 (reference) 1.03 1.13
(H. Yoshikawa,
M. N. Hayashi
& N. Sueoka,
These results and our complete data are summarized in the transfer map shown in Fig. 9, which also includes for comparison the map previously obtained by the markerfrequency method.
SYNCHRONOUS
REPLICATION
OF
361
CHROMOSOME
Marker frequency map? ind his2 fhr
ade 6 I 0
I 0.1
I
I
0.2
fvr
hisf
0.3
0.4
0.5
ode6 ode33 ade34 ode16 /
leyl I
0.6
phe
ileu lysl met I I
ilva
0.7
T
0.8
I.0
0.9
Transfer map ura 1CL)
his1 CL) ode17 sV[ I c OY O”
thr
0.2
leu
met3CL)
0.3
0.4
0.5
06
try2(L)~/ys~i/va’met~//eu
phe (L)
o-7
0.8
I.0
0.9
FIG. 9. Genetic map of B. subtilis. The marker frequency map is constructed from marker-frequency analyses. Original data will be reported later (H. Yoshikawa, R. Neumen & N. Sueoka, manuscript in preparation). The synchro-transfer map is based on analyses shown in Tables 2, 3, 4 and 5. t Summarized by Sueoka, 1966. 5 Data of Oishi & Sueoka (1965), Oishi, Oishi & Sueoke, (1966) and Dubnau, Smith & Marmur (1965) indicate that ribosomal and soluble RNA loci lie between a&l6 and ucZe6. See the transduction data of Anagnostopoulos quoted in the Discussion. The order of these markers cannot, be determined from the transfer experiments.
4. Discussion A simple method has been presented for determining the replication order and relative positions for markers on the B. subtilk chromosome. The results are generally in good agreement with previous results obtained by marker-frequency analysis (Yoshikawa & Sueoka, 1963a). The success of employing transfer experiments in mapping depends on the completeness and maintenance of synchrony of chromosome replication throughout one generation of cell growth. We have found that the best synchrony can be obtained in B. subtilis by germinating microscopically homogeneous spores in a rich medium. This method is particularly useful for markers in the early replicating part of t,he chromosome, where the synchrony of chromosome replication is excellent. However, the method is limited in that synchrony partially deteriorates during the later period of replication (see Appendix). For this reason, in presenting our transfer map in Fig. 9 we have placed the markers tryS(L), lys, &a, met and ileu in the order observed in our transfer data but have not assigned any map positions to them. For the relative map positions of these markers it is necessary to use linkage analyses by transformation or transduction. Such data have been obtained by Anagnostopoulos (1966, personal communication), who has also determined that his ilva, phe and lys markers are very closely linked to our ilva, phe and lys markers, respectively. The order and relative positions of these markers determined by transduction are: try genes lYS I
h-l
met
ilva
ileu
l-l-1personal communication).
(Anagnostopoulos, This is most likely the real order for these markers. Our original assignment of the tryptophan region close to the leu locus has been
362
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N.
SUEOKA
questioned by Kelly & Pritchard (1965) and by Barat, Anagnostopoulos & Schneider (1965). Masters & Pardee (1965) obtained a map position of the try locus closer (0.85) to the terminus than our original value (0.64) by marker-frequency analysis. We have also been informed that the try marker has been located at 0.98 by marker-frequency analysis in Dr Y. Ikeda’s laboratory (J. Nukushina, personal communication). Our present results support the findings of these others who locate try2 close to met. This should remove any difficulty in interpreting the linkage results of Kelly & Pritchard (1965) and Barat et ~2. (1965). Two markers, ileu and met, have been shown to be linked by transformation (Yoshikawa & Sueoka, 1963a). The present methods as well as marker-frequency analysis are suitable for positioning a marker in relation to the entire chromosome. The map thus obtained shows the replication order of markers and defines the replication origin and terminus. For fine mapping within a region, either transformation or transduction linkage experiments should be used. We wish to acknowledge the technical assistance of Mm B. Sesztak. The work was supported by grants from the National Institutes of Health, GM10923-04 and the National Science Foundation, GB3445. REFERENCES Anagnostopoulos, C. & Spizizen, J. (1961). J. Bad. 81, 741. Barat, M., Anagnostopoulos, C. & Schneider, A. M. (1965). J. Bad. 90, 357. Dubnau, D., Smith, I. & Marmur, J. (1966). Proc. Nut. Acad. Sci., Wwh. 54, 724. Kelly, M. S. & Pritchard, R. H. (1965). J. Bact. 89, 1314. Masters, M. & Pardee, A. B. (1966). Proc. Nut. Acad. Sci., Wash. 54, 64. &hi, M., Oishi, A. & Sueoka, N. (1966). Proc. Nut. Acad. Sci., Wash. 55, 1095. &hi, M. & Sueoka, N. (1966). Proc. Nut. Acczd. Sci., Wash. 54, 483. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature, 204, 1069. Sueoka, N. (1966). In Cell Synchrony, ed. by I. L. Cameron & G. M. Padilla, p. 38. New York: Academic Press. Sueoka, N. & Yoshikawa, H. (1965). Genetics, 52, 747. Wake, R. G. (1963). Biochem. Riophya. Res. Comm. 13, 67. Yoshikawa, H. (1965). Proc. Nat. Acd Sci., Wash. 53, 1476. Yoshikawa, H., O’Sullivan, A. & Sueoka, N. (1964). Proc. Nut. Acad. Sci., Wash. 82, 973. Yoshikawa, H. & Sueoka, N. (19630). Proc. Nat. Acad. Sci., Wash. 49, 559. Yoshikawa, H. & Sueoka, N. (19633). Proc. Nut. Acad. Sci., Wash. 49, 806.
APPENDIX
Statistical Analysis A mapping procedure of markers by synchronization of chromosome replication and density-transfer experiments (synchro-transfer analysis), is presented. (a) Basic principles The basic feature of the synchro-transfer experiments is to start with cells the chromosomes of which are in a completed state of replication. Such cells are transferred into a new medium with different isotopes or base analogs which alter the density of newly replicated DNA. Synchronous chromosome replication starts at the time of transfer. For this analysis the mode of replication of the chromosome should be sequential with a fixed order (Fig. 10). For the present analysis, some terminology is given below: x: position of replication point or position on the chromosome (see Sueoka & Yoshikawa, 1965). 2: mean position of x; total length of chromosome is taken as a unit.
SYNCHRONOUS
REPLICATION
FIG. 10. Diagrammatic
OF
representation
363
CHROMOSOME
of necessary
statistics.
X: a genetic marker occupying position X on the chromosome. fractional amounts of transforming activity of marker X,,, X,, and L: non-replicated (oo), half-replicated (on) and fully replicated (nn) peaks in density-gradient. Do,, D,, and D,,,,: Fractional amounts of DNA in non-replicated (oo), replicated (on), and fully replicated (nn) peaks in CsCl density-gradient. 3x: Fraction of chromosomes whose replication point is distal to the marker
1 f(x) = =o
e=p -
(x -2)s 2a2
1 or C(t) = rT
t2
exp - 2-: Gaussian distribution
X in CsCl halfX.
function,
x--z
where t = -.
CJ c: standard deviation of a replication point at certain times after transfer. Diagrammatic representations of these statistics are shown in Fig. 10. Assuming that the distribution of replication point (x) is Gaussian, 1 x (x - 53)”~ Ax = 1 - d(2,)o --m e=p - T. s Physically, the lower limit of x is 0. However, negative values of x do have meaning in the distribution. This point will be explained later. Putting x---z tx=-, (T 5 1 t2 A, =0*5 - ~/(2rr) o exp - 2 dt, (1) s = 0.5 s0
tx d(t) dt,
A. O’SULLIVAN
364
Ol!
‘I+(t)dt=
AND
N.
SUEOKA
105--J.
s 0
Here
t,
represents a
t
value corresponding to X. The relationship
between t and
t 4(t) dt can be obtained from tables of the Gaussian (normal) distribution. s0 t
If A, is available, A, > 0.5,
t,
can be calculated from the table of
4(t) dt. Note that when s we git equations (3). - z . (3) u remains the same in a single
is negative. For two markers, X, and X,, x, t -x1--tx2 = ~ Xl u Since the standard deviation (u) in the above equations DNA sample, x, = (X, - Z) F + 2. (4) Xl The above is the fundamental equation for mapping genetic markers by synchrotransfer experiment. When positions of two markers (reference markers) are known, f can be calculated as: 2s wx, - xztx, (5) tx, - tx1 This allows us to calculate the position of the third marker X, by equation (4) by replacing X, or X, with X, and txl or txa with t,,. z can have negative values for earlier DNA samples and be more than 1 for later samples. These situations are theoretically meaningful and justify the calculation of t,: (1) When 5 < 0, only a fraction of the chromosomes (A,) have initiated their replication (Fig. ll), and this temporal heterogeneity of the initiation itself creates a distribution of the replication point (x). Increased spreading of the distribution in later samples should result from the heterogeneity in the rate of replication. In this connection, the present analysis allows us to calculate u for each sample by using equation (3) (Table 6). (2) When Z > 1 and there is no resting period, the fraction of chromosomes (A,) must have been re-initiated at the origin (Fig. 11). However, as tx
I--AL,----Zt-0
2nd replication
1
1st replication
FIG. 11. Theoretical extension of replication points beyond the origin and terminus. The chromosome occupies the space only between 0 and 1. However, in the early stage of replication where the inception of replication occurs in a fraction of the population, the distribution of z should be treated as extending to negative values (see I). The fraction of chromosomes in the negative area (1 --A,) is in reality not replicating. When the replication approaches the terminus, the distribution should theoretically be extended beyond 1 (see II). In reality, if there is no resting period, the fraction of chromosomes with z= 1 constitutes the second replication point.
SYNCHRONOUS
REPLICATION TABLE
OF
36.5
CHROMOSOME
6
Calculation of %, X and u Sample
1
Marker
adel6 i &UC hisl(L) ade6
4
5
X,, (%I
22,5 33.4 40.3 62.7
77.5 66.6 59.7 37.3
X,,
Average value of 0
A,?
6.x
5s
x
4
0633 0.499 0.425 0.229
- 0.34 0.003 0.19 0.74
0.02 (1st)
0 0.02 0.03 0.07
0.06 0.04 0.07
0.06
ade6 thr
836 85.2 86.6 97.0
16.4 14.8 13.4 3.0
0.195 0.174 0.155 0.031
0.86 0.94 1.02 I.87
0.23 (2nd)
0 0.02 0.04 0.27
0.27 0.27 0.27 0.27
0.27
ade6 thr met3( I,) i leu
65.3 70.5 75.1 836
34.7 29.5 24.9 16.4
0.533 0.418 0.332 0.196
- 0.08 0.21 0.43 0.86
0.16 (2nd)
0.12 0.27 0.39 0.62
0.50 0.52 0.56 0.53
0.53
(leu lY8 ilva i met
56.8 65.6 69.9 71.4
43.2 34.4 30.1 28.6
0.761 0.524 0.431 0.401
- 0.71 - 0.06 0.18 0.25
0.88 (2nd)
0.62 0.86 0.95 1.00
0.37 0.33 0.38 0.48
0.39
udel6 2
X,,
1
8UC
t For sample 1, A, was calculated with equation (7), and for samples 2,4 and 5, A, was calculated with equation (9). $ 5 was calculated by using equation (5) with reference values of X and corresponding t,. (1st) represents the first replication position and (2nd) the second replication position. X-5 5 D was calculated by 0 = ~ for each set oft, and X. Note the estimate is not reliable when tx t, and (X-Z) are close to zero.
far as genetic markers in the terminus region are concerned, further replication by succeeding replication points will not occur unless the synchrony is extremely poor. The z can also be obtained from the distribution of a marker in the oo and on DNA peaks and the distribution of a marker of known position. Such a method should be particularly useful in the early replication stages (D,, = 0), with a known marker close to the origin. The fractional amount of hybrid molecules (Do,) can be equated as:
Therefore
co s
zf(z)dz
0
DO,
= -. 2-
Don
366
A. O’SULLIVAN
AND
N.
SUEOKA
x-2
Since t = -,
u
1 t2 m - - exp - -2 + P 2
11. st,
=U
= u&J
;(Wt
+ tit A,.
Here, to is the t-value corresponding to x = 0, and A,, is the fraction of the chromosomes whose replication has been started (Fig. 11). Since t, = 2, u m
xj(x)dx
= c&t,) + &,A,, = &,
s 0
and
on
DO, u = (2 - Do,) {#(to) - bQ - toDon ’ = - tou = (2 - D,,) {cj(to) - toA,}’
Calculation of tx depends on the situation of chromosome replication, be discussed below.
which will
(b) Replication witA one replication point (i) Within one generation after transfer (X,,, = 0), x,,
= 2 X
or A, =
X 2*-
(7)
By using two genetic markers (X, and X2), corresponding A, and A, can be obtained using equation (7). The values A, and A, allow us to get txl and txl from equation (2). Therefore, if we know the map positions of X, and X2, Z can be obtained from equation (5). This allows us to calculate the positions of unknown markers using equation (4). When an estimate of D,, and the fraction of marker X0 (the marker closest to the origin) in the hybrid region (X,,(O)) are available, map positions of other markers can be calculated from distributions of those markers in parental and hybrid DNA regions. In B. subtilis, marker 0 can be approximated by adel6. Thus, from equation (7) A = &n(O) 0 2 - L(O) using equation (2) and the table of Gaussian distribution, to and $(to) can be obtained. Since the map position of X0 is 0, from equation (4)
x= wot- tx). 0
Here 2 can be obtained from equation (6).
(8)
SYNCHRONOUS
REPLICATION
(ii) In the second generation (X,, = 0, X,, x,,
OF
CHROMOSOME
367
> X,,)
AX = l+A,
Or
A, =
X 1c-y
(9)
Using A, and A, from distributions of two known markers, X, and X,, we obtain txl and txa, and subsequently 2. This allows us to calculate map positions of unknown markers using equation (4). (c) Synchronous replication with two replication position.9 (Fig. 12) Stage 1. The same analysis as in the previous section (first generation) can be applied. Note that stage 1 does not give information on distal markers. Stage 2. (i) Markers which distribute only in oo and on DNA peaks can be analyzed by equation (7). In order to calculate 5, distributions of two markers of known map positions are necessary (use equation (5)). (ii) Markers which are distributed only in on and nn peaks can be analyzed by equation (9) with two reference markers.
00 on nn Stage
0
Stage 1 i
Stage 2 i
Stage
3 i DNA
distribution
FIG. 12. Replication with two replication positions (dichotomous replication). Densities of 00, on. and van DNA’s depend on the kind of isotopes or bese analogs used. Note that the distribution of markers among the DNA peaks is different from marker to marker, depending on their map positions.
368
A. O’SULLIVAN
AND
N. SUEOKA
(iii) If a marker is distributed in all three DNA peaks (00, on, nn), only approximate values can be obtained by assuming either X,, N 0 or X,, ‘v 0. In order to get more accurate values, samples from earlier or later times should be analyzed for DNA after the transfer, so that situation (i) and (ii) can be obtained. Stage 3. In this stage, analysis of distal markers can be made using equation (9) with two reference markers. Markers which are distributed more in the nn peak than in the on peak should not be used for calculation. (d) Notes on application of the theory Map positions of genetic markers have been calculated by the above theory using the experimental data shown in Table 1. The results are given in Tables 2 to 5. Table 6 presents estimates of Z and u calculated from the data of Table 1. The increase of u in time indicates a breaking down of synchrony, which is, in itself, an interesting problem. As far as mapping is concerned, the theory can successfully be applied if distributions of the first and second replication positions do not overlap. When the two distributions overlap as in later stages of the replication, the estimations of 2 and c become unreliable. This situation is evident in the analysis shown in Table 6. The fact that the value of u decreases from sample 4 (O-53) to sample 5 (0.39) indicates the complication created by the overlapping of the first and second replication points. As a convenient guide rule, data which show the value of c as more than half of the distance (d) between the two consecutive replication positions should not be taken seriously. The value of d is 1 for replication with one replication point, and 0.5 for that with two replication positions (Sueoka & Yoshikawa, 1965). This means that the analysis shown in Table 6, where d = 0.5, is reliable for samples 1 and 2, and that data for samples 4 and 5 have only a qualitative value. The extent of the synchrony can conveniently be expressed by u and the corresponding 2, and experimental improvement should be directed toward decreasing the value of (T.The initial value of (Tcomes from the heterogeneity in the initiation of chromosome replication. The increase of 0 in time is due to the heterogeneity in the rate of replication from chromosome to chromosome.
Sequential Replication
of the Bacillus subtilis Chromosome
IV.7 Genetic Mapping by Density Transfer Experiment AIDEEN
O’SULLIVAN
AND NOBORU
SUEOKA
Department of Biology, Princeton University Princeton, New Jersey, U.S.A. (Received 13 December 1966, and in revised form
3 March 1967)
Genetic markers near the replication origin of the chromosome have been mapped in two strains of Bacillus sub&s, W23 and W 168, which differ in replication control. A synchronization of chromosome replication during spore germination was used. Two types of density transfer experiments of spores were performed: one, the germination of deuterium oxide-grown spores in a rich aqueous medium and the other, the germination of spores of a thymine-requiring strain in a medium containing 5-bromouracil. Samples were taken at various times during germination and their lysates were centrifuged in a density gradient. The transfer of genetic markers from non-replicated, half-replicated and fully replicated DNA was followed by transformation assays. A theory of mapping applicable to replication synchronization of the chromosome and density transfer experiments (synchro-transfer analysis) has been developed and applied to the present data with special emphasis on markers close to the replication origin. This method of mapping has been extended to the whole chromosome, and the relative distances between markers was estimated by statistical analysis of the transformation data. There was no difference in the replication origin and in the marker order
between the two strains.
1. Introduction The replication order of markers in the Bacillus subtilis W23 chromosome has been determined by marker frequency analysis in transformation (Yoshikawa & Sueoka, 1963a; Sueoka & Yoshikawa, 1965) and confirmed (Yoshikawa C%Sueoka, 19633) by following the order of transfer of genetic markers from heavy to hybrid density positions when synchronized heavy cells (stationary phase in Da0 medium) of B. subtilis W23 are diluted into a light medium. For reasons discussed in these papers, the most likely representation of the replication order is a linear genetic map, which shows that the chromosome replicates sequentially from the replication origin to the terminus, Using strain W168, the replication order was found to be identical to that of strain W23 when spore DNA of st,rain W168 was taken to represent that of completed chromosomes. However, it was found that in strain W168 the marker ratio did not correspond to that expected for completed chromosomes, implying that stationary cells did not contain completed chromosomes and indicating a difference in control of replication (Yoshikawa, O’Sldlivan & Sueoka, 1964). t fart
III
of this series is Yoshikawa,
O’Sullivan 349
& Sueoka, 1964.
360
A.
O’SULLIVAN
AND
N.
SUEOKA
This report presents a more extensive study of genetic markers near the origin of replication in both strains, and describes a method of chromosome mapping by transfer experiments. Synchronization of chromosome replication during sport: & Sueoka, 1964) germination (Wake, 1963; Yoshikawa et al., 1964; Oishi, Yoshikawa was used. The results present a confirmation and extension of the B. subtilis map, with particular emphasis on markers near the origin of replication.
2. Materials and Methods (a) Strains
Donor DNA for transformation was prepared and 23th~~his (from F. Rothman). Recipient strains for transformation: 168ade33 (Mu33) 108ade34 (Mu34) 108Eeu-adel2 (Mu8u12) 168leu-met-ade6
(MuSLl5u6)
1681e~-met-&e16
(Mu8u5u16)
168leu-met-ode11 168auc-i&met
(from
Ural(L)-hial(L)&y2(L) met3(L)&y2(L) PWL) 168Zeu-met-thr 168lewmet-hia 168&a 168lewmet-ileu 1681ys-phe In order to avoid confusion, denoted as (L). Other mutants
from the following
strains: W168, W23
(MuSu5u17)
Spizizen, met(u5) marker formation by Dr M. Masters) (SB5 from Lederberg) (SB26 from Lederberg) (SB 133 from Lederberg) (MuSu5u5) (Mu8u5u2) (Mu36) (Mu8u5ul) (Mu12u17) mutants isolated in the laboratory were isolated in our laboratory.
introduced
by
of J. Lederberg
trans-
are
(b) Media Medium C (Spizizen minimal salts medium) contains, per liter: 14.0 g KzHPO,; 6.0 g KH,PO,; 2.0 g (NH,),SOI; 1 g trisodium citrate,2H,O; 0.2 g MgSO,,7H,O; 5 g glucose. Medium C+ . Medium C supplemented with 500 mg Casamino acids (Difco Labs); 60 mg L-tryptophan; 60 mg base requirement; 100 mg ammo acid requirement per liter. Medium C++. Medium C supplemented with 100 mg Casamino acids (Difco); 5 mg n-tryptophan; 5 mg L-histidine; 10 mg base requirement per liter; no extra amino acid is added for mutant requirement. Low-phosphate-D20-32P-kxbeled sporulation plates. 22 mg MgSO,; 450 mg (NH&SO,; 1.13 g potato extract (Difco); 10 mg CaCl,; 315 mg KsHPO,; 135 mg KHzPO,; 225 mg (pH 7.0) (all dissolved in DzO and sodium citrate; 8 mc aaP; 1.13 g glucose; 0.05 M-T& dried in vucuo), DzO (99.7%) 225 ml. Penassay germination medium. Antibiotic medium 3 for penicillin assay (Difco), 11.7 g per liter. [3H]Thymine spwdation plates. 3H-labeled spores of 23thy-his were prepared on plates of the following composition: 400 ml. medium C; 40 mg L-histidine; 2 mg thymine; 2 g potato extract (Difco); 16 mg MgCI,; 1.6 mg CaCl,; 16 g agar. After autoclaving, sterile glucose (0.5%) and [3H]thymine (1 mc, specific activity 6.7 c/m-mole) were added. Bromouracil germination medium. Medium C; 2.5 pg thymine; 100 pg n-histidine; 100 pg L-alanine; 500 pg C&amino acids (Difco); 200 pg yeast extract (Difco); 25 pg B-bromouracil (Calbiochem.); 5 mg glucose/ml. DzO sporukztion pkztes. The following were suspended in 10 ml. of D,O, dried in vacua, redissolved in 1 liter distilled DsO, sealed, autoclaved and poured into plastic plates: 14.0 g
SYNCHRONOUS
REPLICATION
OF
CHROMOSOME
351
1.0 g sodium citrate,2HzO; 4 mg M&I,; 6.0 g potato extract (Difco); 50 g glucose, and 40 g/l. agar. Rich low-phosphate 32P-labeled germination medium. 8 g Nutrient broth (Difco); 1.5 g Bacto yeast extract (Difco); 3.5 g NaCl; 1 M-Tris buffer (pH 7~3)~ 50 ml. per 1. 9.8 mc of 3aP (neutralized He3zP0,) was added in 30 ml. of the medium.
6g KzHPO,; 97.6 g MgSO,;
KH,PO,; 40 mg C&l,;
Exp. 1. Replication
2.0 g (NH&SO,;
(c) Method.9 order of adenine markers in atrain W168, by DsO transfer experiment spores were prepared by pre-adapting B. subtilis W168 to growth in
3aP-D,0-labeled D,O medium by serial transfer through 40, 65, 95 and 100% D,O media. An overnight culture of cells was grown in D,O-C+ medium and plated (0.2 n&/plate) on D,0-3aPlabeled sporulation plates. Spores were prepared after 10 days incubation at 37°C. The spores were scraped off the plates, filtered through Whatman no. 1 filter paper, washed twice with sterile distilled water, purified by incubation with egg white lysozyme (Worthington, 1 mg/ml., 50 mm, 37”C), and subsequently with sodium lauryl sulfate (2%, 30 min, 37”C), and finally washed several times with sterile distilled water. The spores were germinated in H,O-Penassay germination medium, cell samples were taken at 33, 43 and 53 min after addition of spores, samples were immediately hilled by heat (60°C, 10 mm), cooled in ice-water, lysed with lysozyme (1 mg/ml., 45 min, 37°C) and sodium lauryl sulfate (2%, 30 min, 37’C). The lysates were centrifuged in a C&l densitygradient (final density 1.70 g/cm3) in Tris (0.01 M)-EDTA (0.001 M) buffer (pH 8.4) for 3 days at 25”C, 35,000 rev./min in SW39 rotor of a Spinco model L ultracentrifuge. Drops were collected from the bottom of the tube, each drop was diluted with 1 ml. sterile SSC (O-15 M-NaCl, 0.015 M-Sodium citrate, pH 7.0). The 32P profile was obtained by removing 0.2 ml. from each tube and assaying for 32P activity by precipitation-in the presence of carrier DNA (200 pg crude salmon sperm DNA/ml.)-with 10% trichloroacetic acid. The precipitate was washed on a membrane filter (coarse, Schleicher & Schuell, Keene, N.H.), dried, and radioactivity counted in a liquid-scintillation counter. Transforming activity was determined by accurately removing 0.1 ml. (or 0.05 ml.) from each tube, adding 1 ml. (or 0.5 ml.) of competent recipient cells and plating on selective media after 40 mm incubation and the addition of DNase. Competent cells were prepared (Anagnostopoulos & Spizizen, 1961) by growing cells in C+ medium until the end of the logarithmic growth phase, diluting l/10 into C++ medium and incubating at 37°C for 90 min. Competent cells were either freshly prepared or stored at tenfold concentration in 50/ glucose at -80°C.
Exp. 2. Replication experiment
order
of
adenine
markers
in
strain
23 by bromouracil
transfer
3H-labeled spores of 23thy-his were prepared by 4 days incubation at 37°C on [3H]thymine-sporulation plates. The spores were purified as described for 3aP-Dz0 spores. The 3H-labeled spores were germinated in bromouracil germination medium. Samples were removed at 45, 52, 55, 70 and 85 mm. Subsequent steps were similar to those described for exp. 1.
Exp. 3. MappirLg of B. subtilis
chromosome
by DzO transfer experiment
D,O-treated spores of WI68 were prepared on D,O sporulation plates. On washing, the crude spores formed three layers. Each fraction was purified separately and tested for germination; the fraction giving the fastest germination curve was used for the experiment. The spores were germinated in rich, low-phosphate medium containing 3zP. 6-ml. samples were taken at 50, 60, 73, 85, 100 and 115 min after addition of spores. Subsequent steps were similar to those described for exp. 1.
3. Results Since there is a difference in the control of chromosome replication between B. subt&s strains W23 and W168, it was of interest to determine whether or not markers near the origin of replication were similar. It has been known that a purine-requiring
352
A.
O’SULLIVAN
AND
N.
SUEOKA
mutant (ade6) occurs within 10% of the replication origin in bot,h strains (Yoshikawa & Sueoka, 1963a). When the density of B. su.bti& DNA was exa.m.ined in a preparotivc: CsCl density-gradient, it was observed that various purinr: markers fill into tfwo density groups (henceforth referred to as groups 1 and 2): indicating that there arc at least two purine regions on the chromosome (O’Sullivan, Yoshikawa & Sucoka., unpublished data). The replication order of various adenine (actually purine) markers has been shown by a D,O-H,O transfer experiment in which D,0-32P-labeled spores of strain W168 were germinated in a light Penassay medium. Samples were taken very shortly after the commencement of DNA replication-previously estimated by [3H]thymine incorporation. (In the germinating spore system it is not necessary to use a thyminerequiring mutant for thymine incorporation.) The replication order of markers was determined by centrifuging the lysed samples in C&I, density 1.70 g/cm3, and testing the heavy (parental) and hybrid (newly replicated) DNA regions for transforming activity for the different markers at increasing times after the start of the DNA replication. The sample in Pig. 1 shows that in strain W168 the markers ade16 and ade17 (group 1 density) are about two-thirds replicated at 53 minutes, whereas the markers ade6 (group 2) and met are still completely in the heavy-density peak. In this experiment, the 32P count was not sufficient to estimate the percentage of DNA which had replicated at this time.
Drop no. FIG. 1. Replication
of various
adenine
markers
in D20
transfer
experiment.
D-32P-labeled spores of WI68 were germinated in light Penassay medium. This sample was does not allow us to estimate taken 53 min after start of germination. The low a2P radioactivity 69 and 70) is not significant’ly the replicated amount of DNA. A small aaP peak (drop numbers larger than the background noise. saP radioactivity (--e--e--). adelG (-x-x-) and utZe17 (group 2); Distribution of markers : mZe6 (-o-o-) (-cl--o-) (group 1); met (-O-O-).
SYNCHRONOUS
REPLICATION
353
OF CHROMOSOME
Figures 2 and 3 show that in strain W23 a similar replication order is obtained when [3H]thymine-labeled spores of 23thy-his are germinated in a medium containing 5-bromouracil. In these experiments, parental DNA is in the light-density band and newly replicated DNA in the hybrid region. Figure 2 shows that the markers adel6 and a&l7 (group 1) are transferred, whereas ade6 and a&34 (group 2) are not. In
Parental
Strain 23
,ade34 -32
3000-
2500-
.E E 9u
zoooade6
,I ISOO-
IOOO-
500 -
0 50
52
FIG. 2. [3H]Thymine-labeled min. 3H radioactivity (-++--). adelS(-a-u-)andadel’l (group 2).
56
60 64 Drop no.
spores of 23th~~ks
(-A-A--)
germinated
6%
72
in 5.bromouracil
0 76
medium
for 50
(group l);ade6(-x-x-)andade34(-O-O-)
this sample, the number of ade16 transformants in the hybrid region is 40% of the total. The proportion of DNA which has replicated can be estimated from the distribution of 3H as approximately 3%. A shoulder seen in both the 3H profile and marker profile of ade6 and ade34 (Figs 2 and 3) deserves comment. This shoulder has been observed in nearly all 5-bromouracil transfer experiments. Moreover, all genetic markers can be found in the shoulder to an approximately equal extent, without
354
A. O’SULLIVAN
AND
N.
SUEOKA
regard to the position of markers on the chromosome. Together with the fact that the density of the shoulder is between the parental and hybrid peaks, the shoulder seems to represent DNA molecules which have undergone repair processes. A similar obscrvation was made by Yoshikawa (1965). In the present calculation of the extent of replication, therefore, shoulder activities have not been included.
r-----l,, Stmin23
Parenta I
6000 -
0
65
70
75.. Drop
FIa. 3. [eH]Thymino-l&&d
spores
of
80
BS
90
no.
23th~~hie germinated
in 5-bromowacil
medium
for
86 min. 3H radioactivity ade6 (-a--@-), fhf (-O-O--).
(--O--O--). c&e34 (-x-x-),
adel2 (-A-A---),
c&e33 (-B-m-)
(all
group
2);
Although synchrony in D,O transfer experiments is considerably better than that in 5-bromouracil, the density difference obtained is not sufficient to separate completely the 3H profiles of heavy and hybrid DNA. Figures 2 and 3 and Table 1 confirm the replication order of markers as being adel6, adel7 (group I), ade6, ade34 (group 2) with adel2, ode33 (group 31) and thr still unreplicated. The results of these experiments show that there are at least two groups of ade mutants, that ode markers in density group 1 are replicated earlier than those in group 2, that both groups are replicated before the thr marker, that the earliest known marker in B. sub&s is adel6, and that this marker is located within 2 or 3% of the origin of replication.
SYNCHRONOUS
REPLICATION TABLE
OF
355
CHROMOSOME
1
Marker distribution in the D,O --f H,O transfer experiment
DNA donor W168
Marker
o/o of total trrtnsformants in?
Total no. of transformants HH(oo)
HL(m)
LL(nn)
Sample 1, 50 min after germination
adel6 8uc(Spizizen) hisI adef3 Ural(L)
366 2459 1846 304 1695
21.3 33.4 40.3 62.7 97.9
73.5 66.6 59.7 37.3 2.1
5.2 0 0 0 0
Sample 2, 60 min after
adelf3 auc(Spizizen) ade6 thr metS(L)
133 1109 1360 97 258
10.1 15.7 22.4 31.2 70.1
752 71.8 67.2 66.7 29.9
14.7 12.5 10.4 2.1 0
Sample 4, 85 min after germination
adel6 suc(Spizizen) hial ade6 thr met3(L) leu PheW Ural(L) w/W)
81’7 2587 1913 102 1071 478 375 4051 2537 2588 832
6.3 12.4 8.4 15.2 6.9 12.6 11.8 11.8 17.8 28.1 33.9
35.1 37.6 44.1 65.4 65.7 65.7 74.3 77.6 81.5 69.7 63.2
58.6 50.0 47.4 29.4 27.5 21.8 13.9 10.6 0.7 2.1 2.9
449 13914 3328 14673 6366 2114 5671 4214 52270 6788
6.5 6.8 12.4 10.7 19.8 11.0 15.9 12.8 17.1 14.2
25.4 40.1 45.0 60.7 43.8 58.4 58.8 63.4 59.2 67.3
68.1 53.1 42.6 38.6 36.4 30.6 25.3 23.7 23.7 18.5
met
Sample 5, 100 min after qermination
de16
his met3 leu
Ural(L) lY8 ilva ww
7ne.t
ileu
t HH refers to the heavy density DNA region, HL to the hybrid density DNA region and LL to the light density DNA region. A more general way of designating parental, hybrid and fully replicated DNA ia shown in parentheses to eliminate the confusion of using different density transfer systems (e.g. D,O+H,O, H,O+D,O, bromouracil+T, T-tbromouracil). 00, Both strands are old (parental). on, One strand is old and the other new (hybrid). nn, Both strands are new (fully replicated).
Since chromosome synchrony was obtained during the germination of D,O-treated spores, this method was used to extend the map of the B. subtilis chromosome. In our germination conditions, re-initiation of daughter replication origins occurs before the first replication point reaches the terminus (dichotomous replication) (Yoshikawa et al., 1964; Oishi et al., 1964). Therefore, transfer of markers both from heavy to hybrid density and from hybrid to light density can be examined. D,O-treated 24
356
A.
O’SULLIVAN
AND
N.
SUEOKA
spores of W168 were germinated in a light, rich, low-phosphate medium containing 32P and samples were taken throughout the course of one generation. The lysed samples were centrifuged with CsCl, the 32P count being used to locate the DNA region. Some examples of the results are shown in Figs 4 to 8. Figure 4 shows the replication order of early markers to be udel6, sue, &l(L) and ade6; Fig. 5 shows the replication order of a&6, thr, met3(L), leu; Fig. 6 shows the later markers leu, Ural(L), phe(L), tryZ(L) and met; Fig. 7 shows the replication order of his, metS(L), leu; Fig. 8 shows that ileu, ilva, met and tryB(L) are close together and that these
Hybrid
Ii
Strain 168
Drop no. Fm. 4. D,O transfer experiment. Replication order of markers is shown to be ode16 (-a-@-), (-u-m-) and ade6 (-O-O--). See also Table 1. (a) Transfer sample 1, 50 min after start of germination. (b) Transfer sample 4, 86 min after start of germination.
8~: (-x-x-),
h&l(L)
SYNCHRONOUS
20
1
I 1 Parenta I
,
,
REPLICATION
1
1
I
OF CHROMOSOME
&.$
18-
’
i
’
’
’
‘Stra’in
16b
Fully replicated
Drop
no.
FIG.6
Replication (-B--B-) germination.
order of markers and Zeu (-A--A-).
FIG. 5. Da0 transfer experiment. is shown to be ade6 (-@-a-), thr (-x-x-), m&3(L) T ransfer sample 4 (same aa Fig. 4(b)), 85 min after start of
FIQ. 6. D20 transfer experiment. Replication order is shown to be Zeu (-A-A-), urul(L) (-x-x-), phe(L) (-O--O-), tryl(L) (-O-O-) and met (-W-m-). Transfer sample 4 (same as Figs 4(b) and 5), 85 min after start of germination. See also Table 1.
358
A. O’SULLIVAN
AND
N.
SUEOKA
18 16
t
G 5 %
-
Strain 168
22-
$
Fully replicated
18-
Parental
l4-
6’-
0
50
55
60
65
Drop no. FIG.8 FIG. 7. D,O transfer experiment. Replication order of markers is his (-O-O-), w&S(L) (-m-w--) Transfer sample 6, 100 min after start of germination. See also Table 1.
and Zeu (-A-A-).
FIG. 8. D20 transfer experiment. Replication order is shown to be urul(L) (-O-O--), ilwa (-x-x--), met (-•-•.r), try!Z(L) (-•--a---) and ileu (-A-A-). Transfer sample 5 (same as Fig. 7), lOOmin start of germination. See also Table 1.
after
SYNCHRONOUS
REPLICATION
OF
359
CHROMOSOME
markers oannot be given their map positions from the results. This is not unexpected, since these are very late markers and synchrony deteriorates toward the end of one generation. The position of the auc marker (0.02) mapped by the present method agrees well with the results (0.013) of Masters BEPardee (1965) by marker-frequency analysis. The results of this experiment are summarized in Table 1, which shows the proportion of each marker in heavy, hybrid and light bands. The replication order, and hence presumably the map order, can be seen to be adel6, SW, hial( adeb, thr, his, met3(L), Zeu,urul(L), phe(L), [ilua, tryS(L), Zya,met, ileu]. The square brackets around the last group of markers indicate that their order is uncertain. In addition to the replication order, the relative distance between markers can be estimated from a statistical analysis of these results (using any one sample) by measuring either the distribution of a marker between heavy and hybrid regions or between hybrid and light regions, and relating it to the distribution of either two known markers or one known marker and the DNA (e.g. from the 32P count). These analyses are shown in Tables 2 to 5. (The method of calculation of the map position X will be found in the Appendix.) TABLE 2 Transfer sample 1 (see Table 1): HH --) HL andyais~ oh of transformsnts involved in heavy to hybrid trensfer which 8re in heavy (HH) and hybrid (HL)
Marker
adel6 auc(Spi2.) hisl(L) ode6
JWXoo)
HWGn)
22.5 33.4 40.3 62.7
77.5 66.6 59.7 37.3
Map position
(X)
O$ (reference marker) 0.02 0.04 0.07-i (reference marker)
t Used when X,, N 0 8nd X,, > 0. For symbols X,, and X0,, see Appendix. $ Adel6 is the closest marker to the replication origin so far obtained. Map position 0 is tern. porarily assigned to this marker. The position of ade6 is the average estim8te m8de by markerfrequency 8nalysis (H. Yoshikawa, M. N. Hayeshi & N. Sueoka, menuscript in preparation).
TABLE
3
Transfer sample 2 (see Table 1): HL + LL analyaes~ Marker
o/0 of transformants in HL end LL regions HWGJ
adel6 suc(Spiz.) de6 thr
83.6 85.2 86.6 97.0
t Not accurate due to the existence of three However, there is residual ctctivity of ads16 from adel6 velues in 18ter samples) which makes the symbols X0,, end X,,. see Appendix. $ An average estimate by merker-frequency Sueoka, mtwiusoript in preparation).
Map position
X
W-L”) 16.4 14.8 13.4 3.0
0 (reference marker) 0.02 0.06 0.271 (reference marker)
pe8ks of tr8nsforming activity (see Table 1). non-repliceting chromosomes of about 7% (see analysis of HL+LL somewhat justifiable. For analysis
(H. Yoshikawa,
M. N. Hayashi
& N.
360
A. O’SULLIVAN
AND
N.
SUEOKA
TABLE 4 Transfer
sample 4 (see Table 1): HH -+ HL and HL + LLT analyses o/0 of transformants
Marker
=Woo)
adel6 suc(Spiz.) on
HWL)
13.6
86.4
17.9 28.7 34.9
82.1 71.3 65.1
in HL and LL regions
HH and HL regions
HWLJ
Map position
(X)
w&n)
34.7 29.5 24.9 16.4 12.0
65.3 70-5 75.1 83.6 88.0
0.11 0.27$ (reference) 0.40 0.623 (reference) 0.78 0.73 0.91 1.003 (reference)
t Used when X,, N 0 and X,. > X,,. The results are not accurate because of the large values of (I. $ Average estimates by marker-frequency analysis (H. Yoshikawa, M. N. Hayashi & N. Sueoka, manuscript in preparation).
TABLED Transfer
Marker
sample 5 (see Table 1): HL + LL analysis o/0 of transformants in HL and LL regions HWLJ
met3(L) leu lY8 ilVlZ
met trYw) ileu
51.4 56.8 65.6 69.9 71.4 72.8 78.4
t Average estimates by marker-frequency manuscript in preparation).
Map position
(X)
w-&J
48.6 43,2 34.4 30.1 28.6 27.2 21.6 analysis
0.26 0.62t (reference) 0.92 0.97 1.007 (reference) 1.03 1.13
(H. Yoshikawa,
M. N. Hayashi
& N. Sueoka,
These results and our complete data are summarized in the transfer map shown in Fig. 9, which also includes for comparison the map previously obtained by the markerfrequency method.
SYNCHRONOUS
REPLICATION
OF
361
CHROMOSOME
Marker frequency map? ind his2 fhr
ade 6 I 0
I 0.1
I
I
0.2
fvr
hisf
0.3
0.4
0.5
ode6 ode33 ade34 ode16 /
leyl I
0.6
phe
ileu lysl met I I
ilva
0.7
T
0.8
I.0
0.9
Transfer map ura 1CL)
his1 CL) ode17 sV[ I c OY O”
thr
0.2
leu
met3CL)
0.3
0.4
0.5
06
try2(L)~/ys~i/va’met~//eu
phe (L)
o-7
0.8
I.0
0.9
FIG. 9. Genetic map of B. subtilis. The marker frequency map is constructed from marker-frequency analyses. Original data will be reported later (H. Yoshikawa, R. Neumen & N. Sueoka, manuscript in preparation). The synchro-transfer map is based on analyses shown in Tables 2, 3, 4 and 5. t Summarized by Sueoka, 1966. 5 Data of Oishi & Sueoka (1965), Oishi, Oishi & Sueoke, (1966) and Dubnau, Smith & Marmur (1965) indicate that ribosomal and soluble RNA loci lie between a&l6 and ucZe6. See the transduction data of Anagnostopoulos quoted in the Discussion. The order of these markers cannot, be determined from the transfer experiments.
4. Discussion A simple method has been presented for determining the replication order and relative positions for markers on the B. subtilk chromosome. The results are generally in good agreement with previous results obtained by marker-frequency analysis (Yoshikawa & Sueoka, 1963a). The success of employing transfer experiments in mapping depends on the completeness and maintenance of synchrony of chromosome replication throughout one generation of cell growth. We have found that the best synchrony can be obtained in B. subtilis by germinating microscopically homogeneous spores in a rich medium. This method is particularly useful for markers in the early replicating part of t,he chromosome, where the synchrony of chromosome replication is excellent. However, the method is limited in that synchrony partially deteriorates during the later period of replication (see Appendix). For this reason, in presenting our transfer map in Fig. 9 we have placed the markers tryS(L), lys, &a, met and ileu in the order observed in our transfer data but have not assigned any map positions to them. For the relative map positions of these markers it is necessary to use linkage analyses by transformation or transduction. Such data have been obtained by Anagnostopoulos (1966, personal communication), who has also determined that his ilva, phe and lys markers are very closely linked to our ilva, phe and lys markers, respectively. The order and relative positions of these markers determined by transduction are: try genes lYS I
h-l
met
ilva
ileu
l-l-1personal communication).
(Anagnostopoulos, This is most likely the real order for these markers. Our original assignment of the tryptophan region close to the leu locus has been
362
A.
O’SULLIVAN
AND
N.
SUEOKA
questioned by Kelly & Pritchard (1965) and by Barat, Anagnostopoulos & Schneider (1965). Masters & Pardee (1965) obtained a map position of the try locus closer (0.85) to the terminus than our original value (0.64) by marker-frequency analysis. We have also been informed that the try marker has been located at 0.98 by marker-frequency analysis in Dr Y. Ikeda’s laboratory (J. Nukushina, personal communication). Our present results support the findings of these others who locate try2 close to met. This should remove any difficulty in interpreting the linkage results of Kelly & Pritchard (1965) and Barat et ~2. (1965). Two markers, ileu and met, have been shown to be linked by transformation (Yoshikawa & Sueoka, 1963a). The present methods as well as marker-frequency analysis are suitable for positioning a marker in relation to the entire chromosome. The map thus obtained shows the replication order of markers and defines the replication origin and terminus. For fine mapping within a region, either transformation or transduction linkage experiments should be used. We wish to acknowledge the technical assistance of Mm B. Sesztak. The work was supported by grants from the National Institutes of Health, GM10923-04 and the National Science Foundation, GB3445. REFERENCES Anagnostopoulos, C. & Spizizen, J. (1961). J. Bad. 81, 741. Barat, M., Anagnostopoulos, C. & Schneider, A. M. (1965). J. Bad. 90, 357. Dubnau, D., Smith, I. & Marmur, J. (1966). Proc. Nut. Acad. Sci., Wwh. 54, 724. Kelly, M. S. & Pritchard, R. H. (1965). J. Bact. 89, 1314. Masters, M. & Pardee, A. B. (1966). Proc. Nut. Acad. Sci., Wash. 54, 64. &hi, M., Oishi, A. & Sueoka, N. (1966). Proc. Nut. Acad. Sci., Wash. 55, 1095. &hi, M. & Sueoka, N. (1966). Proc. Nut. Acczd. Sci., Wash. 54, 483. Oishi, M., Yoshikawa, H. & Sueoka, N. (1964). Nature, 204, 1069. Sueoka, N. (1966). In Cell Synchrony, ed. by I. L. Cameron & G. M. Padilla, p. 38. New York: Academic Press. Sueoka, N. & Yoshikawa, H. (1965). Genetics, 52, 747. Wake, R. G. (1963). Biochem. Riophya. Res. Comm. 13, 67. Yoshikawa, H. (1965). Proc. Nat. Acd Sci., Wash. 53, 1476. Yoshikawa, H., O’Sullivan, A. & Sueoka, N. (1964). Proc. Nut. Acad. Sci., Wash. 82, 973. Yoshikawa, H. & Sueoka, N. (19630). Proc. Nat. Acad. Sci., Wash. 49, 559. Yoshikawa, H. & Sueoka, N. (19633). Proc. Nut. Acad. Sci., Wash. 49, 806.
APPENDIX
Statistical Analysis A mapping procedure of markers by synchronization of chromosome replication and density-transfer experiments (synchro-transfer analysis), is presented. (a) Basic principles The basic feature of the synchro-transfer experiments is to start with cells the chromosomes of which are in a completed state of replication. Such cells are transferred into a new medium with different isotopes or base analogs which alter the density of newly replicated DNA. Synchronous chromosome replication starts at the time of transfer. For this analysis the mode of replication of the chromosome should be sequential with a fixed order (Fig. 10). For the present analysis, some terminology is given below: x: position of replication point or position on the chromosome (see Sueoka & Yoshikawa, 1965). 2: mean position of x; total length of chromosome is taken as a unit.
SYNCHRONOUS
REPLICATION
FIG. 10. Diagrammatic
OF
representation
363
CHROMOSOME
of necessary
statistics.
X: a genetic marker occupying position X on the chromosome. fractional amounts of transforming activity of marker X,,, X,, and L: non-replicated (oo), half-replicated (on) and fully replicated (nn) peaks in density-gradient. Do,, D,, and D,,,,: Fractional amounts of DNA in non-replicated (oo), replicated (on), and fully replicated (nn) peaks in CsCl density-gradient. 3x: Fraction of chromosomes whose replication point is distal to the marker
1 f(x) = =o
e=p -
(x -2)s 2a2
1 or C(t) = rT
t2
exp - 2-: Gaussian distribution
X in CsCl halfX.
function,
x--z
where t = -.
CJ c: standard deviation of a replication point at certain times after transfer. Diagrammatic representations of these statistics are shown in Fig. 10. Assuming that the distribution of replication point (x) is Gaussian, 1 x (x - 53)”~ Ax = 1 - d(2,)o --m e=p - T. s Physically, the lower limit of x is 0. However, negative values of x do have meaning in the distribution. This point will be explained later. Putting x---z tx=-, (T 5 1 t2 A, =0*5 - ~/(2rr) o exp - 2 dt, (1) s = 0.5 s0
tx d(t) dt,
A. O’SULLIVAN
364
Ol!
‘I+(t)dt=
AND
N.
SUEOKA
105--J.
s 0
Here
t,
represents a
t
value corresponding to X. The relationship
between t and
t 4(t) dt can be obtained from tables of the Gaussian (normal) distribution. s0 t
If A, is available, A, > 0.5,
t,
can be calculated from the table of
4(t) dt. Note that when s we git equations (3). - z . (3) u remains the same in a single
is negative. For two markers, X, and X,, x, t -x1--tx2 = ~ Xl u Since the standard deviation (u) in the above equations DNA sample, x, = (X, - Z) F + 2. (4) Xl The above is the fundamental equation for mapping genetic markers by synchrotransfer experiment. When positions of two markers (reference markers) are known, f can be calculated as: 2s wx, - xztx, (5) tx, - tx1 This allows us to calculate the position of the third marker X, by equation (4) by replacing X, or X, with X, and txl or txa with t,,. z can have negative values for earlier DNA samples and be more than 1 for later samples. These situations are theoretically meaningful and justify the calculation of t,: (1) When 5 < 0, only a fraction of the chromosomes (A,) have initiated their replication (Fig. ll), and this temporal heterogeneity of the initiation itself creates a distribution of the replication point (x). Increased spreading of the distribution in later samples should result from the heterogeneity in the rate of replication. In this connection, the present analysis allows us to calculate u for each sample by using equation (3) (Table 6). (2) When Z > 1 and there is no resting period, the fraction of chromosomes (A,) must have been re-initiated at the origin (Fig. 11). However, as tx
I--AL,----Zt-0
2nd replication
1
1st replication
FIG. 11. Theoretical extension of replication points beyond the origin and terminus. The chromosome occupies the space only between 0 and 1. However, in the early stage of replication where the inception of replication occurs in a fraction of the population, the distribution of z should be treated as extending to negative values (see I). The fraction of chromosomes in the negative area (1 --A,) is in reality not replicating. When the replication approaches the terminus, the distribution should theoretically be extended beyond 1 (see II). In reality, if there is no resting period, the fraction of chromosomes with z= 1 constitutes the second replication point.
SYNCHRONOUS
REPLICATION TABLE
OF
36.5
CHROMOSOME
6
Calculation of %, X and u Sample
1
Marker
adel6 i &UC hisl(L) ade6
4
5
X,, (%I
22,5 33.4 40.3 62.7
77.5 66.6 59.7 37.3
X,,
Average value of 0
A,?
6.x
5s
x
4
0633 0.499 0.425 0.229
- 0.34 0.003 0.19 0.74
0.02 (1st)
0 0.02 0.03 0.07
0.06 0.04 0.07
0.06
ade6 thr
836 85.2 86.6 97.0
16.4 14.8 13.4 3.0
0.195 0.174 0.155 0.031
0.86 0.94 1.02 I.87
0.23 (2nd)
0 0.02 0.04 0.27
0.27 0.27 0.27 0.27
0.27
ade6 thr met3( I,) i leu
65.3 70.5 75.1 836
34.7 29.5 24.9 16.4
0.533 0.418 0.332 0.196
- 0.08 0.21 0.43 0.86
0.16 (2nd)
0.12 0.27 0.39 0.62
0.50 0.52 0.56 0.53
0.53
(leu lY8 ilva i met
56.8 65.6 69.9 71.4
43.2 34.4 30.1 28.6
0.761 0.524 0.431 0.401
- 0.71 - 0.06 0.18 0.25
0.88 (2nd)
0.62 0.86 0.95 1.00
0.37 0.33 0.38 0.48
0.39
udel6 2
X,,
1
8UC
t For sample 1, A, was calculated with equation (7), and for samples 2,4 and 5, A, was calculated with equation (9). $ 5 was calculated by using equation (5) with reference values of X and corresponding t,. (1st) represents the first replication position and (2nd) the second replication position. X-5 5 D was calculated by 0 = ~ for each set oft, and X. Note the estimate is not reliable when tx t, and (X-Z) are close to zero.
far as genetic markers in the terminus region are concerned, further replication by succeeding replication points will not occur unless the synchrony is extremely poor. The z can also be obtained from the distribution of a marker in the oo and on DNA peaks and the distribution of a marker of known position. Such a method should be particularly useful in the early replication stages (D,, = 0), with a known marker close to the origin. The fractional amount of hybrid molecules (Do,) can be equated as:
Therefore
co s
zf(z)dz
0
DO,
= -. 2-
Don
366
A. O’SULLIVAN
AND
N.
SUEOKA
x-2
Since t = -,
u
1 t2 m - - exp - -2 + P 2
11. st,
=U
= u&J
;(Wt
+ tit A,.
Here, to is the t-value corresponding to x = 0, and A,, is the fraction of the chromosomes whose replication has been started (Fig. 11). Since t, = 2, u m
xj(x)dx
= c&t,) + &,A,, = &,
s 0
and
on
DO, u = (2 - Do,) {#(to) - bQ - toDon ’ = - tou = (2 - D,,) {cj(to) - toA,}’
Calculation of tx depends on the situation of chromosome replication, be discussed below.
which will
(b) Replication witA one replication point (i) Within one generation after transfer (X,,, = 0), x,,
= 2 X
or A, =
X 2*-
(7)
By using two genetic markers (X, and X2), corresponding A, and A, can be obtained using equation (7). The values A, and A, allow us to get txl and txl from equation (2). Therefore, if we know the map positions of X, and X2, Z can be obtained from equation (5). This allows us to calculate the positions of unknown markers using equation (4). When an estimate of D,, and the fraction of marker X0 (the marker closest to the origin) in the hybrid region (X,,(O)) are available, map positions of other markers can be calculated from distributions of those markers in parental and hybrid DNA regions. In B. subtilis, marker 0 can be approximated by adel6. Thus, from equation (7) A = &n(O) 0 2 - L(O) using equation (2) and the table of Gaussian distribution, to and $(to) can be obtained. Since the map position of X0 is 0, from equation (4)
x= wot- tx). 0
Here 2 can be obtained from equation (6).
(8)
SYNCHRONOUS
REPLICATION
(ii) In the second generation (X,, = 0, X,, x,,
OF
CHROMOSOME
367
> X,,)
AX = l+A,
Or
A, =
X 1c-y
(9)
Using A, and A, from distributions of two known markers, X, and X,, we obtain txl and txa, and subsequently 2. This allows us to calculate map positions of unknown markers using equation (4). (c) Synchronous replication with two replication position.9 (Fig. 12) Stage 1. The same analysis as in the previous section (first generation) can be applied. Note that stage 1 does not give information on distal markers. Stage 2. (i) Markers which distribute only in oo and on DNA peaks can be analyzed by equation (7). In order to calculate 5, distributions of two markers of known map positions are necessary (use equation (5)). (ii) Markers which are distributed only in on and nn peaks can be analyzed by equation (9) with two reference markers.
00 on nn Stage
0
Stage 1 i
Stage 2 i
Stage
3 i DNA
distribution
FIG. 12. Replication with two replication positions (dichotomous replication). Densities of 00, on. and van DNA’s depend on the kind of isotopes or bese analogs used. Note that the distribution of markers among the DNA peaks is different from marker to marker, depending on their map positions.
368
A. O’SULLIVAN
AND
N. SUEOKA
(iii) If a marker is distributed in all three DNA peaks (00, on, nn), only approximate values can be obtained by assuming either X,, N 0 or X,, ‘v 0. In order to get more accurate values, samples from earlier or later times should be analyzed for DNA after the transfer, so that situation (i) and (ii) can be obtained. Stage 3. In this stage, analysis of distal markers can be made using equation (9) with two reference markers. Markers which are distributed more in the nn peak than in the on peak should not be used for calculation. (d) Notes on application of the theory Map positions of genetic markers have been calculated by the above theory using the experimental data shown in Table 1. The results are given in Tables 2 to 5. Table 6 presents estimates of Z and u calculated from the data of Table 1. The increase of u in time indicates a breaking down of synchrony, which is, in itself, an interesting problem. As far as mapping is concerned, the theory can successfully be applied if distributions of the first and second replication positions do not overlap. When the two distributions overlap as in later stages of the replication, the estimations of 2 and c become unreliable. This situation is evident in the analysis shown in Table 6. The fact that the value of u decreases from sample 4 (O-53) to sample 5 (0.39) indicates the complication created by the overlapping of the first and second replication points. As a convenient guide rule, data which show the value of c as more than half of the distance (d) between the two consecutive replication positions should not be taken seriously. The value of d is 1 for replication with one replication point, and 0.5 for that with two replication positions (Sueoka & Yoshikawa, 1965). This means that the analysis shown in Table 6, where d = 0.5, is reliable for samples 1 and 2, and that data for samples 4 and 5 have only a qualitative value. The extent of the synchrony can conveniently be expressed by u and the corresponding 2, and experimental improvement should be directed toward decreasing the value of (T.The initial value of (Tcomes from the heterogeneity in the initiation of chromosome replication. The increase of 0 in time is due to the heterogeneity in the rate of replication from chromosome to chromosome.