Reassociation characteristics of DNA synthesized in wounded pea seedlings

State University of Ghent, Lab. Physiol. Chern. and Lab. Biochemistry, Fac. Medicin and Fac. of Agricultural Sciences, Gent, Belgium

Reassociation Characteristics of DNA Synthesized in Wounded Pea Seedlings D. BROEKAERT and R. VAN PARrIS With 5 figures Received 24 March 1978 . Accepted 14 April 1978

Summary A reassociation study was carried out with DNA isolated from unwounded and wounded pea epicotyl tissues (sheared DNA, mean molecular weight 450,000 d). Spectrophotometric reassociation curves of reference DNA's, of control and wounded tissue DNA, were identical up to Cot 100. In order to compare the reassociation characteristics of newly synthesized DNA, labelled by radioactive precursor incorporation, with the reassociation characteristics of total isolated DNA, the reassociation mixture was fractionated by hydroxylapatite column chromatography. By this technique it was shown that the reassociation of newly synthesized and total DNA coincides during the initial phase (up to Cot 0.1). However, between Cot 0.1 and 1000 labelled DNA reassociated markedly slower than total DNA; a difference of 10 to 15 010 was found. Reassociation was also studied in the presence of an excess of driver DNA, originating from tissues with a varying degree of polyploidy: a similar difference was stated between the reassociation characteristics of total and labelled DNA. This peculiar reassociation is not due to insufficient procedures but represents a real phenomenon, resulting from the underreplication or non-replication of repetitive DNA sequences in cambium nuclei. The main characteristics of reassociated duplexes (density, thermal stability and fragment-length) prove that the reassociation is a reliable reaction. Our results are mainly discussed in relation to the thesis that differential DNA replication is involved in stress-induced DNA synthesis. Key words: wounded peas, DNA synthesis, DNA reassociation, reassociated duplexes, repetitive DNA.

Introduction The crown gall disease is caused by Agrobacterium tumefaciens (SMITH and TOWNSEND) Conn after wounding and infection of susceptible host plants. The main-point in crown gall research concerns the elucidation of the chemical nature of the tumor inducing principle (T.I.P.), the factor postulated to be responsible for the transformation process. These investigations were however extremely controversially and different candidates for the T.!. P. factor were proposed (review by DRLICA and KADO, 1975 and KADO, 1976). After a quater century research, tumor inducing (Ti) Z. Pflanzenphysiol. Rd. 89. S. 169-184. 1978.

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plasmids have been discovered in the inciting bacteria. Actually, these Ti-plasmids seem to be the best candidates for the T.I.P. (KADO, 1976; CHILTON et a!., 1977). Our main interest in the crown gall transformation process was restricted to the problem of the induction of DNA synthesis by sterile wounding of host plants. DNA synthesis induced by wounding has been studied previously in Vicia faba stem segments (KUPILA and STERN, 1961; KUPILA and THERMAN, 1971; RASCH, 1964), Kalanchoe stem segments (LIpETz, 1967) and potato tuber tissues (WATANABE and IMASEKI, 1973). However, the characteristics of this wound tissue DNA synthesis were studied in situ or by elementary chemical dosage techniques; DNA was not isolated and characterized. GUILd: et a!. (1968) were the first to isolate and characterize double-stranded wound tissue DNA. Afterwards, these authors suggested that the synthesis, after wounding, of a specific nuclear GC-rich satellite DNA is an obligatory step involved in the initial transformation process (GUILLE and QUETIER, 1970). Our data concerning DNA synthesis in decapitated pea seedlings unequivocally established that DNA synthesized after wounding belongs to the main-band DNA class (BROEKAERT and VAN PARIIS, 1975). At that time we stressed that synthesis of nuclear satellite DNA, after wounding plant tissues, is certainly not a general phenomenon; the main causes of this discrepancy were discussed (BROEKAERT and VAN PARIIS, 1975). Meanwhile we made an autoradiographic study on the induction of DNA synthesis in wounded pea seedlings (BROEKAERT and VAN PARIIS, 1977), in combination with an extensive biochemical study of the base composition of wound tissue DNA (BROEKAERT and VAN PARIIS, 1978). These investigations supported the conclusion that the newly synthesized DNA has to be considered as the product of the genome replication during the Svphase of a newly induced mitotic and/or endomitotic cell cycle, mainly in (pro)cambium nuclei in the immediate vicinity of the wound region; only a minor number of cortex nuclei (2 C, 4 C) is involved. It was our intention to complete the characterization of the DNA synthesis phenomenon in wounded tissues. We focussed our investigations on the reassociation characteristics of the newly synthesized and of the total DNA in wounded and control pea seedlings. The reassociation was studied using the spectrophotometric reassociation technique and the hydroxylapatite (HAP) column fractionation technique in different circumstances. Furthermore, the validity of the experiments was tested: reassociated DNA duplexes were isolated and their elementary characteristics were compared with those of native DNA. Materials and Methods The plant material, condition of germination, wounding and [6- 3H] or [2- 14 C] thymidine reeding, DNA isolation and preparative CsCI gradient centrifugation procedures were as outlined before (BROEKAERT and VAN PARIJS, 1978). Reference DNA or DNA added in excess in some reassociation experiments was obtained from chromatin isolated following the method of COUCKE and VAN PARIJS (1972). DNA was prepared from pure chromatin by a

Z. Pflanzenphysiol. Ed. 89. S. 169-184. 1978.

Reassociation characteristics of DNA

171

four- to sixfold deproteinization step (BROEKAERT and VAN PARIJS, 1978) and only DNA spooled on a glass rod after addition of 2.5 vol ethanol was collected and purified finally by RNase-treatment (Ribonuclease A, bovine-pancreas, type I-A, Sigma) and pronase-treatment (Pronase B grade, Calbiochem). Afterwards, the isolation procedure of chromatin was shortened in order to avoid proteolytic degradation of histones (COUCKE and VAN PARIJS, 1973): rough chromatin was washed four times with 0.14 M NaCl, pH 7.0 and the sucrose gradient centrifugation was omitted. Chromatin proteins were extracted with 2 M NaCI, 5 M urea and chromatin DNA was collected by centrifugation (48 h, 4 DC, 8 X 50 ml Aluminium Angle Rotor MSE, 100,000 g). The DNA pellet was resolved in 0.1 SSC (0.015 M NaCl, 0.0015 M sodium citrate, pH 7.0 ± 0.2) and further purified as described. Spectrophotometric analysis of the reassociation process and the study of the thermal stability of the reassociated duplexes, after thermal denaturation and the T m determination of native DNA were performed as described previously (BROEKAERT and VAN PARIJS, 1978). The reassociation was followed as the decrease in UV absorption at 260 nm in 0.12 M phosphate buffer (PB), pH 6.8 or in 1 SSC at 62 DC using a Gilford 2400 spectrophotometer connected with a Haake circulating waterbath. The reassociation temperature was nearly 20 to 25 DC under the T m of pea DNA in 1 SSC or 0.12 M PB, corresponding to the optimum reassociation temperature (WETMUR and DAVIDSON, 1968). The thermal stability of reassociated duplexes at Cot 100 was determined as described before (BROEKAERT and VAN PARIJS, 1978) and the LITm' the difference between the T m of native and reassociated DNA, was calculated. LlTm provides an indication of the precision of nucleotide pairing (BRITTEN et a1., 1974). MCCARTHY and FARQUHAR (1972) calculated a mean factor, taken over here, for the conversion of .d T m in terms of base mispairing: 1 010 base-mismatching causes a LlTm of 1.5 to 1.6 DC. Furthermore, the thermal stability depends on DNA chain length; this effect appears to be less important in normal experimental procedures and is inferior to the effect of nucleotide mismatching (ULLMAN and MCCARTHY, 1973 a, b). In our work, this effect is of no importance since DNA molecules of comparable size were analyzed. In order to compare the reassociation characteristics of newly synthesized (labelled) DNA with those of total isolated DNA, the reaction mixture was deposited on a hydroxylapatite (HAP) column (HAP, Biogel-H'I', Bio-rad). After extensive dialysis against 0.12 M PB and shearing by an ultrasonic power treatment (BROEKAERT and VAN PARIJS, 1978), the DNA sample was denatured (10 min at 102 Dq, immediately cooled in an ice-bath, diluted with Lidistilled water to an end-concentration of 0.03 M PB and passed over a 10 ern" HAP column at 62 DC. The column was washed intensively with 0.03 M PB and single-stranded DNA was eluted with 0.12 M PB and incubated for reassociation (62 DC, 0.3 M NaCl, 0.1 0/0 sodium lauryl sulphate). This procedure has been shortened mainly because a small fraction of the pea genome reassociated instantaneously and was lost from the 0.12 M fraction. To avoid this loss of «zero time binding» DNA the solution of single-stranded DNA was immediately brought at the final NaCl and SDS concentration, warmed up to 62 DC and incubated for reassociation.

At different Cot values a sample of the reassociation solution was analyzed on a HAP column at 62 DC following the method of MCCALLUM and WALKER (1967): single-stranded DNA was eluted with 0.12 M PB, partially reassociated DNA and fully reassociated DNA were eluted with 0.16 and 0.30 M PB respectively. The elution peaks were fractionated and each fraction was tested for UV absorption (260 nm) and radioactivity incorporation (BROEKAERT and VAN PARIJS, 1975). The percentage of reassociation was calculated after correction for the hypochromiciry (+ 200(0) of reassociated DNA. Originally, all Cot values were calculated as the product of Co (molar concentration of nucleotides) and t (time in seconds). Co values were obtained from the absorption measurements at 260 nm and were in agreement with the values calculated from the Z. Pflanzenphysiol. Bd. 89. S.

169~184.

1978.

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diphenylamine reaction (BURTON, 1956) and from the 268.5 nm absorption after 5 Ofo TCA hydrolysis (15 min at 90 DC), using the molar absorption coefficient of LOGAN er al. (1952). OD Routinely however, Cut values were calculated from the formula : ~ X t, with t time in hours and OD 26o the optical density of the single-stranded DNA solution (BRITTEN and KOHNE, 1966). The thermal stability of isolated duplexes was determined by spectrophotometric analysis or HAP chromatography, the density was investigated by preparative CsCI gradient centrifugation (BROEKAERT and VAN PARIJS, 1978). In order to study to molecular weight of reassociated duplexes, the 0.3 M fraction of the final HAP fractionation was dialysed against 0.2 SSC at 4 DC and precipitated with 2.5 vol ethanol (-20 DC, 2 h) after addition of Na-acetate to a final concentration of 0.3 M. The precipitated DN A was collected by centrifugation (15 min, SS34 Servall rotor, 12,000 rpm, ODC) and concentrated after dissolving in 0.2 SSC (neutral sucrose gradient) or in 0.7 M NaCI, 0.3 M NaOH, 0.01 M Tris, 0.001 M EDTA (alcaline sucrose gradient). The profile of sedimentation was analyzed after preparative sucrose gradient centrifugation (5-20 Ofo w lv, 19 h, 24,000 rpm, SW 25-1 rotor Spinco, 4 DC) and compared with the sedimentation profile of sheared DNA (4.5 X 105 dalton ). Reassociated duplexes in 0.2 SSC were also denatured (10 min, 102 DC) in the presence of 4 010 formaldehyde and analyzed by sucrose gradient centrifugation in the presence of formaldehyde to prevent reassociation.

Results Sp ectrophotometric analy sis

0/ the reassociation process

The reassoc iation of reference DNA's (intact 3-d ay-old seedlings, 5-day-old plumula and epicotyl tissues), isolat ed from chromatin and shea red by an ultrasonic power treatment, was followed in 0.12 M PB pH 6.8 at 62 DC (Fig. 1 a). Th e re association curves, recorded up to C ot 100, are id entical : a n instantaneously reassociating fr action was det ected in all samples and at C ot 0. 1, 1, 10 and 100, the reassociat ion percentages are respec ti vely 10, 22-24, 39 and 43-46 0/0. Beyond Cot 50, the th ree reas sociation curves bend , indic ating the end of reassociation of a sepa ra te DNA class. The reassociat ion of 1- and 2-d a y-old wound tissue DNA (epicotyl 12 d) and control DNA fr om unwounded epic ory l tissues (12 d) w as ana lyzed in the same w ay (Fig. 1 b). Again the re association curves show no difference and at Cot 0.1, 1, 10 and 100 respectively 10, 22.5 , 35 and 42 Ofo of the tot al DNA was reassociated. From th ese experiments we conclude that no clear differences are found in total DNA isolated from unwounded and wounded tissues.

HAP analysis

0/ the

reassociation process

Initially, reassociation experiments were restricted to the analysis of sheared DNA, isolated from wounded a nd unwounded epicot yl tissues, at C ot 1 and / o r Cot 10. The reas socia tion percentage of the newl y synt hesized (ra dioactive) DNA wa s always 10 to 15 % retarded in compar ison w ith the rea ssociation percentage of total DNA (260 nm absorption) (T able 1). Th ese specific rea ssociation charact eri stics we re observed in more th an 20 indep end ent exp eriments a nd neith er feeding wi th Z. Pjlanzenphysiol. Bd. 89. S. 169- 184. 1978.

Reassociation characteristics of DNA

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Fig. 1: Spectrophotometric registration of the reassociation of sheared DNA at 62 DC in 0.12 M PB, pH 6.8. a) 3-day-old seedling DNA (e), 5-day-old plumula DNA (X) and epicotyl DNA (0) was isolated via chromatin; only DNA spooled on a glass rod was involved. b) 12-day-old intact epicotyl DNA (e), 1-day-old wound tissue DNA (0) and 2-day-old wound tissue (X) DNA, isolated by the modified STERN (1968) procedure (BROEKAERT and VAN PARrJs, 1978) and purified by preparative CsCI gradient centrifugation. 12-day-old seedlings were wounded by decapitation. different precursors ([6- 3H ] TdR or [2_14C] TdR), wounding young (3-6 d) or older (8-9 d) seedlings, studying the reassociation in optimal (62 DC) or stringent (72 DC) conditions, nor isolation of DNA by different methods, could substantially effect this typical reassociation pattern. These results were also not affected by a different efficiency of radioactivity countings in different solvents. In the first experiments, as stated in «Materials and Methods», the «zero time binding» DNA was not involved in the reassociation mixture (Fig. 2). However, when this instantaneously reassociating DNA was present in the reassociation mixture, the differentiated reassociation characteristics of labelled and total DNA were still observed. The average length of the sheared DNA fragments affects the reassociation rate as appears from the mathematical description of the reassociation kinetics (BRITTEN and KOHNE, 1966) and from initial (MARMUR and DoTY, 1961; MARMUR er al., 1963; BRITTEN and KOHNE, 1966) and more extended experiments by WETMUR and DAVIDSON (1968). Consequently, for a clear experimentation, the fragment Z. Pflanzenphysiol. Rd. 89. S. 169-184. 1978.

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Table 1: Reassociation of sheared DNA isolated from control and wounded epicory ls (8 d): comparison of the reassociation of total DNA (E 260 ) and newly synthesized DNA (cpm). Fraction (in

0/ 0 )

0.12 M E 260 cpm

0.16 M E 260 cpm

0.30 M E 260 cpm

54.2 35.5 27.1

57.2 42.0 34.8

3.9 4.0 4.1

12.8 12.2 14.0

41.9 60.5 68.7

30.0 45.8 51.2

88.5 92.1 74.3 75.4 62.0 66.6 44.0 54.5 44.3 56.1 45.9 58.1 40.0 47.0 32.8 44.8

4.2 9.9 0.5 3.3 0.7 1.7 1.0 4.0

1.1 6.6 4.0 4.8 3.6 2.9 3.0 3.4

7.3 15.8 37.5 52.7 55.0 52.4 59.0 63.2

6.8 18.0 29.4 40.6 40.3 39.0 50.0 51.6

l -carrier DNA 100-carrier DNA 0.1 + seedling DNA (3 d) 1 + seedling DNA (3 d) 10 + seedling DNA (3 d) 1 + plum . DNA (5 d) 1 + epic. DNA (5 d)

40.7 49.5 22.3 35.6 76.2 70.1 47.0 54.0 39.7 42.0 45.6 53.2 41.7 52.4

8.2 4.2 2.8 7.0 5.8 10.0 7.0

6.6 4.4 8.6 9.6 8.6 10.5 7.7

51.1 43.9 73.5 60.0 21.0 21.3 46.0 36.3 54.5 48.3 44.4 36.1 51.3 39.8

4. Wou/ld tissues 3 d Cot I-carrier DNA Cot 100-carrier DNA C ot 1 + seedling DNA (3 d) Cot 10 + seedling DNA (3 d) Cot 1 + plum. DN A (5 d) Cot 1 + epic. D N A (5 d)

52.2 56.2 23.5 36.0 47.1 50.7 39.1 45.4 45.7 52.3 39.2 50.7

5.7 7.8 4.6 2.5 6.3 9.3

5.8 5.0 9.5 7.7 11.3 10.0

42.1 37.8 68.7 59.0 48.3 39.6 58.4 46.9 48.0 36.4 51.5 39.3

Stage

1. Unwounded epicotyls Cot 0.5 Cot 5 Cut 51 2. Wound tissues 1 d Cot 0.05-carr ier D N A Cot O.l -car rier DNA Cot 0.8 + seedling DNA (3 d) Cot 1 + seedling DNA (3d) Cot 11 + seedling DN A (3 d) Cot 150 + seedling DNA (3 d) Cot 960 + seedling DNA (3 d) Cot 1250 + seedli ng DNA (3 d) 3. Wound tissues 2 d

Cot Cot Cot Cot Cot Cot Cot

DNA fro m unwounded and wounded tissues was isolat ed following the metho d of STERN (1968), modified by BROEKAERT and VAN PARIJS (1978) and purified by preparative CsCI gradient cent rif ugation. Carrier D NA was isolated from chromatin (COUCKE and VAN PARIJS, 1973). Reassociati on of shear ed DNA was carried out in 0.12 M PB, p H 6.8, 0.3 M NaCl, 0.1 Ofo SDS at 62 °C. Ana lysis of the reassociati on mixture was carried out by H AP chromatography at 62 °C.

length of the compared DNA samples has to be identical. In our experimental conditions, this demand is fulfill ed both for unsheared (mean molecular weight 3. 106 d) and sheared (450,000 d) DNA. However, one must realize that a distinct fluct uation of the chain length ar ound an average value exists in unsheared DN A ; Z. Pjlanzenphysiol. Bd. 89. S. 169-/84. 1978.

Reassociation characteristics of DNA

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Fig. 2: Analysis of the reassociation mixture on HAP at 72 "C. Wound tissue DNA, isolated from 3-day-old wounded tissues (seedling 3 d) was purified following MACGILLIVRAY et al. (1971) and denatured (10 min, 102 DC). Single-stranded DNA was isolated by HAPchromatography at 72 DC (a). At Cot 1 after reassociation in 0.12 M PB, 0.3 M NaCl, 0.1 0/0 SDS at 72 DC, the reassociation mixture was analysed on a water-jacketed HAP column at 72 DC (b). Symbols: UV absorption at 260 nm (0), incorporated radioactivity (e).

clearly, this fluctuation is decreased by intensive shearing as demonstrated by sucrose gradient centrifugation or polyacrylamid gel electrophoresis (not represented). Within one experiment, in a strict sense, the chain length of unlabelled and labelled DNA must be identical too, a demand completely fulfilled in our case as demonstrated in Fig. 3. From this type of experiments we conclude that the different reassociation behaviour of labelled and unlabelled DNA is not caused by a different chain length of reassociating strands. We and other research-workers regularely observed fluctuations of the reassociation percentage at comparable Cot values in different experiments; consequently, the results must be interpreted with sufficient caution. These fluctuations, among other things, are mainly caused by the use of HAP batches Z. Pjlanzenphysiol. Bd. 89. S. 169-184. 1978.

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Fig. 3: Analysis of the fragment length of sheared DNA from 3-day-old wounded tissues in an alcaline sucrose gradient (5-20 Ofo w/v, 4 DC, 19 h, 23,000 rpm, SW 25-2, Spinco). a) wound tissue DNA in the presence of carrier DNA isolated from cotyledons, b) wound tissue DNA without carrier DNA. 8-day-old seedlings were wounded by decapitation. Symbols are explained in the legend of Fig. 2. originating from different production series and by structural changes of HAP during successive fractionations, resulting in other binding characteristics (BRITTEN et al.,1974). Our results confirm the statement that reassociation of sheared DNA, investigated by the HAP technique, precedes the reassociation followed optically: the effect is nearly 20 % in the middle part of reassociation curves and is mainly due to the fact that spectrophotometric analysis gives a direct estimate of the fraction involved in nucleotide pairing, while by the HAP technique, double-stranded DNA containing a variable amount of single-strands is isolated (BRITTEN and KOHNE, 1966; LAIRD, 1971). Furthermore, the addition of NaCI (0.3 M) in the reassociation mixture, analyzed afterwards by HAP fractionation, not only causes an increase of the optimal reassociation temperature, but also of the reassociation rate (GILLIS et al., 1970). The use of very young seedlings in investigations concerning DNA synthesis induced by wounding is disadvantageous since DNA replication associated with polyploidisation of cortex nuclei may interfere with the stress-induced DNA replication. This interference is minimal in older seedlings, used by preference. More complete information concerning the reassociation characteristics was drawn by investigating early (e.g. Cot 0.1) and late (e.g. Cot 1000) points of the reassociation process. For practical reasons the DNA concentration was augmented by addition of a 10 to lOa-fold excess of carrier DNA, originating from chromatin of intact seedlings (3 d). The results are also summarized in Table 1 and are interpreted Z. Pjlanzenphysiol. Bd. 89. S. 169-184. 1978.

Reassociation characteristics of DNA

177

as follows. At Cot values less than or equal to Cot 0.1, the same reassociation percentage for total and labelled DNA was observed. Between Cot 1 and 100 the reassociation percentage of labelled DNA is again 8 to 13 % lower than the yield for total DNA. Analysis of the reassociation mixture at higher Cot values (nearly Cot 1000) demonstrates that the reassociation curves of unlabelled and labelled DNA rather converge. Finally, reassociation experiments in which an excess of plumula (5 d) or epicotyl (5 d) DNA was hybridized against labelled wound tissue DNA prove that also this carrier DNA, can not drive the reassociation of labelled DNA, so that the difference in reassociation percentage between labelled and total DNA disappears (Table 1). Characterization of reassociated duplexes

The density of reassociated DNA, isolated at Cot 2 after reassociation of DNA originating from unwounded and wounded epicotyls, equals 1.701 g/cm" in a neutral CsCl gradient (Fig. 4) and is 0.006 g/cm 3 higher than the native main-band DNA density (BROEKAERT and VAN PARIJS, 1978). The equilibrium density profile of reassociated duplexes originating from sheared DNA is slightly asymmetric towards a

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Fig. 4: Preparative CsCI-gradient centrifugation of isolated repetitive DNA (Cot 2.25), after reassociation in 0.12 M PB, pH 6.8, 0.3 M NaC!, 0.1 % SDS, at 62°C and HAP chromatography at 62°C. a, b) analysis of repetitive DNA from unwounded epicotyls, in the presence (b) or absence (a) of marker DNA (<5 = 1.695 g/cm 3 ) ; c, d) analysis of repetitive DNA from 3-day-old wounded tissues in the presence (d) or absence (c) of marker DNA (<5 = 1.695 g!cm 3) . 8-day-old seedlings were wounded by decapitation. Symbols are explained in the legend of Fig. 2. Z. Pjlanzenphysiol. Rd. 89. S. 169-184. 1978.

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D. BROEKAERT and R. VAN PARljS

the heavy side, suggesting the presence of single-stranded DNA in the reassociated duplexes rather than a heterogenious base distribution. In the presence of marker DNA, the profile of labelled reassociated DNA shows a shoulder in accordance with the main-band top fraction. This shoulder peak may be an artefact caused by an unspecific interaction of main-band DNA and reassociated duplexes. Analysis of Cot 0.1, 1 and 10 duplexes, after reassociation of 2-day-old wound tissue DNA in the presence of an excess of seedling (3 d) DNA, reveals identical features (not represented) . Numerical data concerning the melting curves of reassociated duplexes (Cot 100) after reassociation of reference DNA's, and DNA isolated from wounded and control tissues in the spectrophotometer, are presented in Table 2: reassociated duplexes have a T m value between 74.80 and 75.60 oC, nearly 8°C lower than sheared native DNA and 11°C lower than unsheared native DNA (BROEKAERT and VAN PARIJS, 1978). LlTm values suggest 5 to 7 % base-mispairing (MCCARTHY and FARQUHAR,1972). Table 2: Thermal denaturation and spectrophotometric T m determination of reassociated duplexes at Cot 100.

DNA isolation

Tm

LIT

01

or

Hyperchromicity

COlo) a) seedling 3 d plumula 5 d epicotyl S d b) epic. 12 d, unwounded epic. 12 d, wounded 1 d epic. 12 d, wounded 2 d

75.5 75.5 75.5 74.8 75.6 75.0

15.2 7.3 16.2 7.1 16.0 7.5 15.2 7.0 14.0 7.0 15.4 7.8

7.9 17.5 9.1 21.0 3.5 19.5 8.2 18.0 7.0 16.5 7.6 16.6

Sheared DNA was reassociated in 0.12 M PB, pH 6.3 at 62°C. a) DNA isolated from chromatin (COUCKE and VAN PARljS, 1973); b) DNA isolated by the modified method of STERN (1963) described by BROEKAERT and VAN PARljS (1973). DNA was precipitated by addition of 2.5 vol ethanol, spooled on a glass rod and finally purified by preparative CsCI gradient centrifugation.

Reassociated DNA, isolated by the HAP technique at Cot 72, after reassociation of sheared DNA in 0.12 M PB, 0.3 M NaCI and 0.1 % SDS at 62 and 72 °C respectively, was melted in a Gilford 2400 spectrophotometer (Table 3): a melting point of 77.0 to 77.8 °C was found after reassociation in mild conditions and 78.4 to 78.8 °C after reassociation in more stringent conditions, suggesting a more accurate nucleotide pairing. The hyperchromicity reaches 23.5 to 27.5 % and is higher than the hyperchromicity of duplexes at Cot 100 (Table 2). Reassociation in stringent conditions, also results in a 3 to 5 % lower reassociati-on percentage in comparison with reassociation at 62°C; a similar situation is described by RANJEKAR and Z. Pjlanzenphysiol. Bd. 89. S. 169-184. 1978.

Reassociation characteristics of DNA

179

Table 3: Thermal denaturation and T m determination of reassociated duplexes, isolated by HAP chromatography, after reassociation of sheared DNA. Tm

LIT

01

or

Hyperchromici ty (010)

- reassociation at 62 DC

77.0 77.8

18.6 19.8

10.0 10.8

8.6 9.0

25.3 27.5

- reassociation at 72 DC

78.4 78.6 78.8

15.6 14.4 15.0

8.2 8.2 8.0

7.4 6.2 7.0

23.7 23.5 23.7

- (1) 3-day-old wound tissue DNA without carrier DNA with seedling (3 d) DNA with plumula (5 d) DNA

77.5 77.5 77.0

13.5 17.0 18.5

6.75 9.50 9.75

6.75 7.50 8.75

- (2) 2-day-old wound tissue DNA with seedling (3 d) DNA with plumula (5 d) DNA

77.5 78.0

14.0 13.0

7.00 6.25

7.00 6.75

DNA isolation

a) Spectrophotometric analysis o] seedling (3 d) chromatin DNA at Cot 72

b) HAP analysis oj Cot 1 DNA':-)

,:-) DNA was isolated by the modified STERN (1968) procedure described by BROEKAERT and VAN PARIJS (1978) and purified by preparative CsCI gradient centrifugation. Carrier DNA was isolated from chromatin (COUCKE and VAN PARIJS, 1973). Reassociation was occurring at 62 DC in 0.12 M PB, pH 6.8, 0.3 M NaC!, 0.1010 SDS; HAP fractionation at 62 DC. (1) 8-day-old seedlings, (2) 4-day-old seedlings were decapitated.

MURTHY (1973) for rat DNA. Analysis of the thermal stability of labelled, isolated

reassociated duplexes (Cot 1) after reassociation in the absence or presence of different carrier DNA's reveals a comparable melting behaviour (Table 3): the T m values are 77.0 to 78.0 DC, 7.0 to 7.5 DC lower than the T m of sheared native labelled DNA (BROEKAERT and VAN PARrIS, 1978), again suggesting 5 0/ 0 mismatching. Finally we investigated the molecular weight of reassociated duplexes (Cot 1) after reassociation in high or low concentration (respectively 2.0 and 0.2 OD 26o) of sheared 2-day-old wound tissue DNA (seedling 4 d), in the absence or presence of carrier DNA (plumula 5 d). One relevant experiment is represented in Fig. 5. Nearly 30 to 40 % of the reassociated DNA has a molecular weight equal to or slightly higher than the mean molecular weight of sheared reference DNA (450,000 d), 60 to 70 % of the duplex DNA bands near the bottom of the gradient and has a high molecular weight, due to branched network formation (concatemers), In any experiment, the specific activity of concatemer DNA was inferior to the specific Z. Pjlanzenphysiol. Bd. 89. S. 169-184. 1978.

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E260..------------------....,....-----, 1-200 a

0800

cpm

2000

1000

lS00

1000

SOO

4

8

12

16

20

traction number

Fig. 5: Analy sis of the molecular weight of reassociated dupl exes by sucrose gradient centr ifugatio n: (a) reference sheared DNA (450,000 d) in an alcaline gradient ; (b) repetitive DNA (Cot 1) in an alcaline gradient or in a neutral gradient (c). DNA was isolated from 2-d ay-old wounded tissues (seedling 4 d), purified by preparative CsCI gradient centrifugation and sheared. Reassociation was carr ied out in 0.12 M PB, pH 6.8, 0.3 M NaCI, 0.1 Ufo SDS at 62 ec in low concent ration in the presence of driver DNA (plumula 5 d). Sucrose grad ient centrifugation in 5-20 Ufo sucrose (w I v ), at 4 ec, dur ing 19 h, 24,000 rpm, SW 25-1 (Spineo). Symbols are explained in th e legend of Fig. 2.

activity of short duple x DNA, suggesting th at unreplicated DNA was, by preference, involved in concatemer networks and contr ibuted to a lesser degree to short duplexe s. Denaturation of reassociated DNA combined by alealine sucrose gradient Z. Pjlanzenpbysiol. Ed. 89. S. 169-1 84. 1978.

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centrifugation reveals one peak in a position fully comparable with the single-stranded sheared DNA position. However, in some experiments we observed that concatemer DNA was not completely converted into single-stranded DNA and still occupied a sedimentation position beyond that of single-stranded sheared DNA. The imperfect separation in an alcaline medium of complementary strands of high molecular weight DNA was mentioned before (JOLLEY and ORMEROD, 1974 and cited authors) and remains a serious problem to rule out,

Discussion From our previous investigations (BROEKAERT and VAN PARIJS, 1978) and the results presented here, we first of all deduced a model for the kinetic complexity of the Pisum sativum genome, presented and discussed in an accompanying short communication. We restrict the discussion here to the main-point, namely the fact that newly synthesized DNA in wounded and control tissues shows reassociation characteristics deviating from those of total isolated DNA. As already mentioned, this departure is not due to insufficient procedures and the analysis of isolated reassociated duplexes points to the same conclusion. Consequently, we suspect that this finding reflects a real phenomen-on. The essence of the reassociation studies demonstrates that 1. reassociation of labelled and total DNA proceeds in a parallel way up to Cot 0.1, and that 2. between Cot 0.1 and 1000, the reassociation of labelled DNA is retarded (10-15 0/0) as compared to the reassociation of total DNA. In terms of kinetic complexity, we state that labelled DNA contains less repetitive sequences, belonging to the heterogenious repetitive DNA, than total DNA. The mean repetition factor of this heterogenious repetitive DNA in peas was estimated to be 3700 (BRO£KAERT and VAN PARIJS, in the accompanying communication). In other words, the ultrasonic power treatment, resulting in a mean fragment length of 750 np (np: nucleotide pairs), liberates more fragments (belonging to this heterogenious repetitive DNA) from the total DNA type than from the labelled DNA type. Consequently, the concentration of the fragments under discussion is smaller for labelled DNA than for total DNA, resulting in well differentiated reassociation characteristics. From histoautoradiographic investigations (BROEKAERT and VAN PARIJS, 1977) it was concluded that 80 % of the nuclei involved in the DNA synthesis phenomenon in wounded pea seedlings are localized in the cambial region in the wound zone, 20 % of the labelled nuclei belong to the cortex. Also in unwounded elongated pea epicotyl tissues almost all labelled nuclei were found in the cambial region. In an elongated pea epicotyl, nearly 60 vascular cells are present per 100 parenchyma cells (VAN OOSTVELDT and VAN PARIJS, 1975). Taking into account the DNA content of procambium and cortex nuclei (BROEKAERT and VAN PARIJS, 1973; 1976), we calculated that maximum 20 to 25 % of the total isolated DNA and nearly 80 Ofo of Z. Pjlanzenphysiol. Rd. 89. S. 169-184. 1978.

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the labelled DNA originates from procambium nuclei. Consequently, by comparing the characteristics of labelled and total DNA, essentially procarnbium- and cortex DNA are compared. This specific distribution of labelled nuclei has no influence on the reassociation characteristics, if the genome of procambium nuclei (2 C or 4 C) is identical to the genome of c-ort ex nuclei (4 C or 8 C). If this supposition is right we may conclude that a fraction of the hererogenious repetitive DNA is underreplicated or not repl icated at all in procambial nuclei after wounding but also in elongated control pea seedlings. In a more general way , we conclude that the specific reassociation data reveals a differential DNA repl ication (discussed further on ). H owever, other possibilities have to be considered. If, due to differential replication during embryogenesis, the procambial genome differs qualitatively from the parenchymal genome, one may ask again whether the cambial genome replication is complete after wounding, or not. Finally, we want to str ess that we obtained preliminary data favouring the hypothesis that a different organisation of the repetitive and unique sequences in labelled and unlabelled DNA can explain the typical reassociation characteristics. Even so, a different genome organisation combined with a different kinetic complexity of total and labelled DNA can be the underl ying phenomenon causing the peculiar reassociation data (BROEKAERT, 1976). A definite and justified choise between the cited, rather speculative statements, can not be made actually. We mad e however an effort to compare the different possibilities . If qualitative differences exist between the genome of a diploid or tetraploid cambium cell and a tetraploid or octaploid cortex cell, the procambium DNA, representing the more «meristernatic» DNA from the epicotyl, may show a greater similarity to DNA isolated from predominantly diploid plumula cells. Our data summarized in Table 1 pro ve that in the presence of an excess plumula DNA (5 d) the difference in reassociation percentage between tot al and labelled DNA is not disappearing. In control experiments, when an excess of other reference DNA is used originating either from seedling 3 d or epicotyl 5 d and thus mainly representing DNA from differentiated polyploid cortex cells, also the different reassociation percent age persists. This is in accordance with the fact th at 1. spectrophotometric analysis of the reassociation of DNA of intact seedlings, epicotyls and plurnula's reveals coinciding reassociation curves (Fig. 1 a) and 2. heteroduplex thermal stability is fully comparable with homoduplex thermal stability (Table 3 b). Consequently, the homolo gy between labelled wound tissue DNA and plumula DNA is of the same order as the homology between labelled wound tissue DNA and epicotyl or seedling (3 d) DNA. The thermal stability of reassociated duplexes is definitely not influenced by a possible lack of repetitive sequences. The fraction of procambium nuclei in an intact seedling, and in a 5-da y-old plumula and epicotyl, is nearly comparable with the fraction in the wounded tissues. From these data, we conclude that the key to these peculiar reassociation data seems to be the fact that labelled DNA is procambial in nature, rather than the fact that it originates from 2-C and 4-C nuclei. Z. Pjlanz enphysiol. Bd. 89. S. 169-184. 1978.

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In the last decennium, new insights in plant genome structure and organisation also led to a better insight in the DNA replication phenomenon itself. Before 1970, a complete DNA replication in S-phase nuclei and subsequently, a perfect doubling of the DNA amount in premitotic nuclei and perfect DNA distribution among daughter nuclei, was accepted. Since that time, differential DNA replication (selective amplification as well as underreplication) was recognized as an effective mechanism governing differentiation (extensive review by NAGL, 1976). Also in Pisum sativum, differential replication should be connected with the differentiation process: underreplication of repetitive DNA in epicotyl parenchyma cells during endomitosis has been suggested (VAN OOSTVELDT and VAN PARIJS, 1976). In diploid epicotyl cortex nuclei, a fraction belonging to the repetitive DNA seems to be absent and thus, was not replicated during subsequent polyploidisation; on the other hand, diploid plumula nuclei should contain this specific DNA fraction. Possibly the missing DNA is synthesized, later on in octaploid cortex nuclei (VAN OOSTVELDT and VAN PARIJS, 1976), as deduced from intensive histophotometric measurements. Up to now, this delayed DNA synthesis in polyploid cortex nuclei was not demonstrated by radioactive precursor incorporation, the most powerful technique in this respect. At the moment, it is not clear to us, whether an analogous phenomenon is involved in the stress induced cambium DNA synthesis. Data from literature concerning this aspect of wound induced DNA synthesis are not available and up to now we were unable to demonstrate the delayed synthesis of repetitive cambium DNA. The possibility that rRNA cistrons were part of underreplicated repetitive DNA has been tested. The high multiplicity of rRNA genes was deduced from hybridization data of CHIPCHASE and BIRNSTIEL (1963) and SINCLAIR and BROWN (1971). The haploid pea genome contains 3900 genes for rRNA (INGLE and SINCLAIR, 1972). This multiplicity corresponds to the mean repetition factor of the heterogenious repetitive DNA involved in our experiments. Investigations, based on liquid DNA-rRNA hybridization-reassociation, using the technique of KOHNE (1968), successful for bacterial genome analysis, did not led to definite conclusions mainly because «zero time binding» DNA, in fact self-reannealing DNA (palindromes), interfered in DNA-rRNA duplex formation. Acknowledgement We wish to thank Dr. P. COUCKE for advice concerning the chromatin isolation and Dr. P. VAN OOSTVELDT for advice and profound discussion concerning the reassociation experiments.

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D. BROEKAERT, State University of Gh ent , Lab . Ph ysio!. Chern. and Lab. Biochemis try, Fac . Me dicin and Fac. of Ag ri cultu ral Sciences, 35, Ledeganckstra ar, B-9 000 Gent , Belgium.

Z. Pflanzenphysiol. Ed. 89. S. 169-184. 1978.