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Caryologia: InternationalJournal of Cytology,Cytosystematics andCytogeneticsPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/tcar20

Restriction Endonuclease (Re-)Banding of Plant ChromosomesIngo Schuberta

a Zentralinstitut fuer Genetik undKulturpflanzenforschung, DDR-4325 Gatersleben,German Democratic Republic.Published online: 30 Jan 2014.

To cite this article: Ingo Schubert (1990) Restriction Endonuclease (Re-) Banding ofPlant Chromosomes, Caryologia: International Journal of Cytology, Cytosystematicsand Cytogenetics, 43:2, 117-130, DOI: 10.1080/00087114.1990.10796991

To link to this article: http://dx.doi.org/10.1080/00087114.1990.10796991

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CARYOLOGIA Vol. 43, n. 2: 117-130, 1990

RESTRICTION ENDONUCLEASE (RE-) BANDING OF PLANT CHROMOSOMES

INGO SCHUBERT Zentralinstitut fuer Genetik und Kulturpflanzenforschung, DDR-4325 Gatersleben, German Demo­cratic Republic.

SUMMARY - The ability of 19 restrictases to produce Re-bands detectable by Giemsa stain in plant chromosomes was tested with Vicia faba, Hordeum vulgare, and Allium cepa. For comparison, some of the restrictases were also used to digest human chromosomes. Additionally, DNasel treatments were performed for uniform and nearly complete digestion of nuclear DNA. DNA extraction by some endonucleases from 14C-labelled nuclei was tested measuring radioactivity of enzyme solutions after treatment of chromosomes. Especially effective in Re-banding of Vicia faba chromo­somes were sequential digestions with Alul -+ Mbol and Ddei -+ Clai. Some restrictases did not produce any bands. However, occasionally control treatments with incubation buffers also resulted in dark Giemsa bands after long treatment times. The applicability of Re-banding for plant chromosomes is dicussed.

INTRODUCTION

The possibility to discriminate between individual (pairs of) chromosomes is a prerequisite for many cytogenetic and genetic studies. Therefore, especially in species possessing chromosomes of similar morphology, longitudinal differ­entiation of metaphase chromosomes is a main goal of cytogeneticists. This is achieved mainly by various banding techniques, by differential incorporation of base analogues, or by in situ hybridization (for review see ScHUBERT and RIEGER 1989). One of the most recent variants of banding (Re-banding) is based on restriction endonuclease treatment prior to Giemsa or fluorochrome staining. This type of banding became adapted to a wide range of species (Tab. 3). It causes differentiation patterns in mammalian chromosomes which resem­ble G- or C-banding. For plants up to now true chromosomal bands obtained after restrictase treatment were reported only in the case of Vicia faba (FRE­DIANI et al. 1987; ScHUBERT and RIEGER 1989).

In this paper we report on results of treatment of metaphase chromosomes from different plant species with 19 restrictases. For comparison, DNasei treatment was performed since it digests chromosomal DNA in situ without

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118 SCHUBERT

producing banded chromosomes (SCHWEIZER 1977; SAHASRABUDDHE et al. 1978). In order to prove enzymatic activity of restrictases against chromosomes in situ, human chromosomes of PHA stimulated lymphocytes were digested by enzymes known to produce a definite Re-banding pattern.

MATERIAL AND METHODS

Squash preparations were made from root tip meristems of a reconstructed karyotype (ACB) of the broad bean Vicia faba with six easily distinguishable pairs of chromosomes (for description see MICHAELIS and RIEGER 1971; DOEBEL et al. 1978), of barley (Hordeum vulgare), and of onion (Allium cepa). Prior to fixation in 3:1 ethanol/a­cetic acid, metaphases were enriched by immersion in colchicine solution (0.05%, or 0.025% in a saturated solution of alpha-bromonaphthalene in the case of barley) for 3 hours.

Subsequently, root tips of V. faba and A. cepa were digested at 3 7 °C in 1% cellulase for 90 minutes and in 2% pectinase for 2h. Both enzymes (from Serva) were diluted in 0.01 M sodium citrate buffer, pH 4.5-4.8. After digestion, root tips were squashed in 45% acetic acid according to the dry ice method, washed in 96% ethanol, and air dried. Slides were stored in glycerol up to several months. Before restrictase treatment, glycerol was washed out by 2 X sse (0.3M NaClf0.03M Na-citrate) for 15 minutes and air-dried slides were equilibrated for 1h in the corresponding incubation buffers. Then 20!!1 drops of buffer and buffer containing enzyme, respectively, were put on the slides. Treatment was performed in moist chambers at 37°C (or 45°C for Aval, II) for 2-24h. Thereafter, the drops were removed by pipettes; slides were shortly rinsed in distilled water and stained in 4% Giemsa (Merck) for 2-5 minutes, air-dried, and mounted in entellan (Merck). Vicia faba root tips were additionally incubated in aerated solutions of 14C-thymidine for 20h before immersion in colchi­cine. After preparation, control slides were autoradiographed to prove incorporation of 14C-thymidine. From slides with 14C-labelled nuclei, the drops of enzyme and buffer were measured after treatment for the degree of DNA extraction in a liquid scintilla­tion counter. For comparison, additional20!!1 drops of buffer plus the scraped off cells covered by buffer drops were measured for estimation of total radioactivity of nuclear DNA.

For preparation of lymphocytes, fresh blood was mixed 1: 1 with Hanks balanced salt solution (Staatliches Institut fuer Immunpraeparate und Naehrmedien, Berlin); 4ml blood on 3ml Ficoll gradients (1.077 g/cm3

) were centrifuged for 30 minutes at 2200 rpm. Lymphocytes were 3 X washed in 8ml Eagle-MEM (Staatliches Institut fuer Immunpraeparate und Naehrmedien, Berlin). Then 2ml RPMI-medium (Staatliches Institut fuer Immunpraeparate und Naehrmedien, Berlin) containing 15% fetal calf serum and 0.05ml phytohemagglutinine were added to the sediment; 1ml of the mixture was pipetted in glass bottles. After cultivation for 69h, 0.015ml colchicine (0.025%) was added for 3h, followed by treatment in hypotonic KCl (0.075M) for 15 minutes. After recentrifugation (10'), the pellet was fixed in 3:1 methanol: acetic acid for 1 day (at the beginning fixative was changed twice), and lymphocytes were dropped on slides and air-dried.

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Re·BANDING OF PLANT CHROMOSOMES 119

The producers of the restriction endonucleases were: Dr. W. Baudisch, ZIGuK, Gatersleben, for Alul, Asul and III, Aval and II, BamHI, EcoRI and V, and Sphl; Boehringer Mannheim, for Clal, Ddel, Hindiii, Rsal and Sail; ZIMET, Jena, for EcoRII, Hinfl, Pvuii and Haeiii; Dr. Rebentisch, NVII-Genetika, Moscow, for Mbol.

The composition of incubation buffers was as recommended by the producers of the enzymes.

RESULTS

1) DNasei digestion.

It was calculated that 1U of DNasei from bovine pancreas (Serva) per slide (diluted in EcoRI incubation buffer) should be sufficient to digest all the DNA of one slide (10,000-30,000 nuclei) if this DNA would be accessible in purified state in solution, since V. faba has a 2C DNA content of < 30pg. 4U DNase/slide in EcoRI buffer led to a nearly complete digestion of the DNA of chromosomes and nuclei from broad bean, onion, and human chromosomes after 2h of treatment. This became evident from the uniform loss of stainabi­lity by Giemsa stain of chromosomal material, while nuclei and chromosomes surrounding the treated area and those covered by buffer drops stained deeply. The same is proved additionally by the high level of soluble radioactivity in the drops containing DNasei. In all DNasei treatments of 14C-labelled V. faba nuclei, significant extraction of radioactivity (1011-3544dpm) was measured. Buffer drops yielded only slightly more radioactivity (96-186dpm) than the background ( <50dpm), and buffer drops containing the scraped off nuclei showed 3844 and 4366dpm, respectively. Though these values are only semi­quantitative, because of varying numbers of nuclei in different areas of individ­ual slides, the tendency confirms an effective digestion of nuclear DNA by DNasei. No chromosomal banding was detectable after DNasei treatment of the three species tested under the conditions described. This agrees with data reported by SCHWEIZER (1977) and SAHASRABUDDHE et a/. (1978).

2) Digestion with restrictases.

As reported for other species (for recent reviews see BIANCHI and BIANCHI 1987 and BABU 1988), not all of the restrictases used resulted in bands on metaphase chromosomes and in pronounced chromocenters in interphase nu­clei (Tab. 1).

a) Vicia faba.

Asui and III, Avail, BamHI, EcoRII, Hindiii, Hinfl, Pvuii, and Sall did not result in chromosome bands detectable by Giemsa stain.

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120 SCHUBERT

TABLE - <<in situ» digestion of chromosomes with restriction endonucleases (treatment conditions, efficiency in Re-banding, and elution of DNA).

Chromosome Elution of

Enzyme Recognition Duration Units/ banding 1

radioactivity sequence (h) slide Vf Ac Hv Hs

(dmp)'

Alul AG!CT 16 20 + + + 1682 A sui G!GNCC 2 2 41 Asuiii GPu!CGPyC 2 2 24 A val GPyCGPuG 16 10 + +' 416

A Avail G!G(T)CC 2 8 150 BamHI G!GATCC 3-24 30 Clal ATtCGAT 17 90 + + 7138 Ddel C!TNAG 17 5-10 + + 7262 EcoRI G!AATTC 2-19 10-50 +' +

A EcoRII !C*C(T)GG 2 3 74 EcoRV GAT!ATC 16 8-100 +' 3356 Haeiii GG!CC 2-16 80 + + 374 Hindiii MAGCTT 2 24 177 Hin£1 G!ANTC 3-24 30 Mbol !GATC 3-16 30 + + 6430 Pvull CAGKTG 3-24 30 Rsal GnAc 16 10 + + 907 Sail G!TCGAC 17 11 Sphl GCATG!C 16 20 + 2967

Vf= Vicia faba; Ac=Allium cepa; Hv=Hordeum vulgare; Hs=Homo sapiens.

1 In all cases of Re-banding on V. faba chromosomes, at least occasionally, bands occurred in buffer controls also.

2 Measured for V. faba only. ' Telomeric bands only in two out of four experiments. ' Bands only after 19h of treatment. ' In V. faba bands only with 100 units/slide; human chromosomes completely digested.

Alul, Aval, Clal, Ddel, EcoRI and V, Haelll, Mbol, Rsal and Sphl resulted in more or less clear bands at chromosomal positions which show up with dark bands also after urea-trypsine or Ba(OH)2 treatment prior to Giemsa staining (DoEBEL et al. 1978). All of these enzymes extracted significant amounts of radioactivity from labelled nuclei (Tab. 1). As in the case of conventional Giemsa banding not all of the faint bands appeared in every celL Most frequently the so-called 'marker bands' (DOEBEL et al. 1978) showed up, especially in segments 23, 26, 4, and 6, followed by bands in segments 22, 27, 15, 14, 19, 18, 10, 11, and 13. Contrary to conventional Giemsa banding, no bands occurred at the nucleolus organizing secondary constriction (segment 2)

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Re-BANDING OF PLANT CHROMOSOMES 121

TABLE 2 - Sequential <<in sitU>> digestion of <Nicia faba» chromosomes by restrictases.

Treatment conditions Chromosome banding'

Elution of radioactivity (dpm)

Alul, lh, 20U Aval, 2.5h, lOU Ddel, 1.5h, 7U EcoRI, 2h, 50U Hindiii, 2h, 24U

--+ Mbol, 16h, 30U --+ EcoRV, 16h, 8U --+ Clal, 15.5h, 90U --+ Avail, 2h, 8U --+ EcoRII, 2h, 3U

+

+

13.082

12,450 150

3,244

' In all cases of Re-banding, at least occasionally, bands occurred in buffer controls also. 2 Complete digestion of chromatin. For further explanation see text.

and at the distal end of the satellite (segment 1) after treatment with different restrictases.

The results obtained with Alul, BamHI, and Mbol correspond to those reported by FREDIANI et al. (1987).

Most pronounced banding patterns, with up to 20 bands per haploid chromosome complement, were obtained after sequential digestion with Alul --.. Mbol and Ddel --.. Clal (Fig. 1).

Sequential digestion with Aval --.. EcoRV, EcoRI--.. Avail, and Hindlll --.. EcoRIII did not result in bands, although not only Alul --.. Mbol and Ddel --.clal but also Hindlll --.. EcoRII eluted very high amounts of radioactivity from labelled nuclei (Tab. 2).

Possibly, the recognition sites of Hindlll and EcoRII are evenly distribut­ed over the chromosomes.

Sequential treatment with Aval --.. EcoRV resulted in unstained ghost­like nuclei and chromosomes. Though elution of radioactivity was not tested in this case, it seems that EcoRV additionally digested the DNA of those regions which appeared as bands after treatment with only Aval.

Restrictase treatment as short as 2h did in no case result in Re-bands. This is also true in the case of sequential digestion with EcoRI --.. Avail.

Remarkably, in some of the experiments resulting in Re-banded chromo­somes, bands occurred after treatment with the corresponding buffers too, though the same buffers did not digest purified lambda-DNA (W. BAumscH, pers. comm.).

b) Allium cepa.

From the restrictases tested in this species, Alul, Asul and III, Avail, Clal, EcoRII, Haelll, Hindlll, and Mbol produced non Re-bands. Digestion with Aval provoked in two out of four experiments dark bands, preferentially at heterochromatic telomeres; digestion with EcoRI produced bands at the

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Re-BANDING OF PLANT CHROMOSOMES 123

centromeres in one experiment and in another not (Fig. 2). IKUSHIMA (1983) reported on chromosome fragmentation after treatment of A. cepa chromo­somes with EcoRI in vivo (3U/J.tl, 0.5h, 37°C).

c) Hordeum vulgare.

Treatment of barley chromosomes with Alui and Clai resulted in weak bands surrounding the centromeres as do conventional Giemsa bands (Fig. 3). Asui and III, Avai, EcoRII, Hindiii, and Mboi did not produce any bands, although SuBRAHMANYAM et al. (1976) observed fragmentation of barley chro­mosomes after in vivo treatment with Hindii and III.

d) Homo sapiens.

In control experiments with human chromosomes from PHA stimulated lymphocytes, C-bands were obtained after treatment with Alui and Ddel (Fig. 4).

Treatment with Ddel, EcoRV, and Mboi for as long as 16h led to nearly complete digestion of nuclear DNA. This was inferred from the unstained, ghost-like appearance of nuclei and chromosomes after treatment. Incubation in EcoRV and Mboi buffers for 16h provoked the appearance of bands in many metaphase chromosomes (Fig. 4).

Contrary to the data of other authors, Haeiii produced weak C-instaed C + G-bands while Mboi and Rsai yielded G-instead of the expected C-like bands.

Avai and Clai did not produce Re-bands. For Avai this result aggrees with the data reported by MILLER et al. (1983).

DISCUSSION

Concerning the mechanisms of Re-banding two possibilities were dis­cussed previously: 1) extraction of short sequences up to a few hundred base pairs of length; 2) distortion of condensed chromosome structure without significant loss of DNA (MEZZANOTTE et al. 1985a, 1985b). Recently however, there is a growing number of arguments suggesting DNA extraction in situ to be the most probable reason for Re-banding (MILLER et al. 1984; BIANCHI and BIANCHI 1987; BABU 1988). Our results of DNA extraction from radioactively labelled nuclei by DNasei and restrictases support this inference. It should however be mentioned that these data are of semiquantitative character, and varying amounts of nuclei covered by enzyme or buffer solutions and/or occasional loss of nuclei from the slides may lead to too low levels of radioacti­vity in enzyme drops and to too high ones in buffers, respectively.

The failure to produce Re-bands by some restrictases might be due either

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124 SCHUBERT

Fig. 2. - Re-bands on metaphase chromosomes of Allium cepa, a) after Aval (20U, 16h, 45°C) preferentially involving telomeric heterochromatin; and b) after EcoRI (lOU, 19h) preferentially involving centromeric heterochromatin. The bar represents 5 j.Lm.

Fig. 3. - Re-bands on metaphase chromosomes of a) Vicia faba, reconstructed karyotype ACB, after EcoRI (lOU, 19h); b) Hordeum vulgare after Clal (90U, 17h).

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Re-BANDING OF PLANT CHROMOSOMES 125

Fig. 4.- Human metaphase chromosomes a) Re-banding after treatment with Alul (20U, 16h); b) Re­banding after Ddei (22U, 3h); c); d); and e) after treatment with Mho incubation buffer for 16h. The bar represents 5 ~tm.

to too long distances between recognition sites, thus preventing the elution of DNA, or to an equal distribution of recognition sites resulting in an equal loss of DNA along the metaphase chromosomes. Confirmation of these ideas comes from improved Re-band resolution by sequential digestion with two restrictases (UEDA et al. 1987 and this paper) and from the fact that short restriction fragments of Vicia faba DNA digested with BamHI (which is unable to produce Re-bands in the same species) evenly hybridized along the chromosome comple­ment (SCHUBERT and RIEGER, in press).

Since the first report on Re-bands in Indian muntjac chromosomes by LIMA DE FARIA et al. (1980), it was shown for many species (Tab. 3) that

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TABLE 3 - Compilation of species investigated for the occurrence of Re-banding after treatment with restriction endonucleases.

Species

Primates: Homo sapiens

Pan troglodytes

Pan paniscus

Gorilla gorilla

Pongo pygmaeus

Macaca fascicularis M. nemestrina M. mulatta M. actroides M. tonkeana

Callithrix ;acchus

Cercopithecus aethiops

Other mammals: Muntiacus munt;ak

Cricetulus griseus

Akodon sp.

Mus musculus

Enzymes Re-bands

Alul, EcoRI", II Ddel, Haeiii, Hindlll", Hinfl, Hpall", Mbol, Rsal

Alul, Ddel, Haeiii, Mbol

Alul

Alul, Ddel, Haeiii, Hin£1, Mbol, Rsal

Alul, Ddel, Haeiii, Hin£1, Mbol

Haeiii

Alul, EcoRII, Haeiii, Hin£1, Mbol, Rsal

Alul, Haeiii

Alul, Haeiii, Mbol, Rsal

Alul, Avail, BamHI, BstNI, EcoRI, II Haeiii, Hindiii, Hin£1, Mbol

No bands References

A val, II, Ball, BamHI, BIANCHI Bgll, EstEll, BstNI, et a!. (1985b) Cfol, Clal, FnuDII, MEZZANOTTE Hhal, Hind, Kpnl, et a!. (1983, Mspl, Pvul, II, Sacl, 1985a) Smal, Sstl, Taql, Xbal MILLER et a!.

(1983); this paper

Hin£1, Rsal

Rsal

Alul Alul Alul Alul Alul

Alul

BIANCHI et a/. (1985a) FERRUCCI eta/. (1987)

FERRUCCI eta/. (1987)

BIANCHI et a/. (1985a) FERRUCCI eta/. (1987)

BIANCHI eta/. (1985a) FERRUCCI eta/. (1987)

DESTEFANO and FERRUCCI (1986)

BIANCHI and BIANCHI (1987)

BamHI, EcorRI, Hhal, BABU and Hindiii, Hpall, Mspl, VERMA (1986) Thai LIMA DE FARIA

et a!. (1980) UEDA et a!. (1987)

BIANCHI and BIANCIII (1987)

BIANCHI and BIANCHI (1987)

ADOLPH (1988) KAELBLING et al. (1984) MEZZANOTTE and FERRUCCI (1984)

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Species

Birds: Gallus domesticus

Aphibia: Bufo americanus

Bufo marinus

Enzymes Re-bands

Haeiii

Haeiii, Mspl, Hindiii, Hpall

Haeiii, Hindiii

Gastrotheca riobambae Haelll, Mspl

Triturus alpestris EcoRI, Haelll, Mspl

Fishes: Muraena helena

Salmo gairdneri

Insects: Arcyptera tornosi (Orthoptera)

Aedes albopictus (Diptera)

Culiseta longiareolata (Dipt.)

Drosophila melanog. (Dipt.)

Sacrophaga bullata (Dipt.)

Plants: Allium cepa

Hordeum vulgare

Vicia faba

Ddel, Haeiii, Mbol

Alul, Haeiii, Hinfl, Mbol, Pvull

Alul, Haeiii, Hinfl

Hpall, Mbol

Alul, BamHI, Ddel, Haeiii, Hhal, Hinfl, Hpall, Mbol, Mspl

Alul, Haeiii

Alul, Haeiii, Hindiii

Aval b, EcoRI b

Alul, Clal

Alul, Aval, Clal, Ddel, EcoRI, V C, Haeiii, Mbol, Rsal, Sphl

a) conflicting data reported, b) not observed in all experiments, c) only at high concentration (100U) of enzyme.

No bands

EcoRI, Hindiii, Hpall, Mspl

Hpall, Mspl

References

ScHMID and DE ALMEIDA (1988)

ScHMID and DE ALMEIDA (1988)

CAu et at. (1988)

EcoRI, Hindiii, Hpal, LLOYD and Mspl THORGAARD (1988)

Hindiii

Hhal, HinPI, Mspl Taql

Alul, Asul, III, Avail, Clal, EcoRII Haeiii, Hindiii, Mbol, Sail

Asul, III, Aval, II, Ddel, EcoRI, II, Hindiii, Mbol, Sail

Asul, III, Avail, BamHI, EcoRII, Hindiii, Hinfl, Pvull, Sail

GosAL VEZ et al. (1987)

BIANCHI et a/. (1986)

MARCHI and MEZZANOTTE (1988)

MEZZANOTTE (1986)

BuLTMANN and MEZZANOTTE (1987)

this paper

this paper

FREDIANI et al. (1987) ScHUBERT and RIEGER (in press) this paper

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128 SCHUBERT

restrictase treatment in situ can be a very distinct means to characterize individual chromosomes by positive (dark) and negative bands (gaps).

The results of FREDIANI et al. (1987) and those reported in this and in a previous (ScHUBERT and RIEGER, in press) paper indicate that the same might be true for plant chromosomes, although, till now, no negative bands, compara­ble to those reported for human chromosomes after digestion with Hinfl (MILLER et al. 1983; BIANCHI et al. 1985b) were observed.

The occasional occurrence of bands in buffer controls on Vicia, human (and mouse, ScHUBERT unpublished) chromosomes which was also observed by BIANCHI et al. (1985b) needs an explanation. Possibly, long time exposure (> 16h) of chromosomes to buffers at 37° or 45°C represents a borderline condition for changing chromosome structure in a way that Giemsa bands may arise, which thus may be confused with true Re-bands. After treatment of Vicia chromosomes for 6h with Ddel and Dde buffer, respectively, the enzyme yielded clear Re-bands, while in buffer control nearly no bands appeared.

CONCLUSION

Although Re-banding in V. faba yielded with two exceptions in the same banding patterns as conventional Giemsa banding, Re-bands were often less clear. Application of restrictases is more expensive and more time consuming than the Ba(OH)2 method. Therefore, Re-banding may be useful for studying specific questions, but not for routine chromosome banding in plants.

The occurrence of bands after long incubation in buffer demands careful controls to avoid a mis-interpretation of data.

Acknowledgements. - I am thankful to Dr. G. KUENZEL for preparation of barley chromo­somes, to Miss U. MERGNER for preparation of human chromosomes, to Dr. W. BAUDISCH, B. HoFEMEISTER, and Prof. U. WOBUS for providing samples of restrictases, to Dr. D. HELLMUND for purified DNasel, to Andrea NERLICH and Barbara HILDEBRANDT for skilfull technical assistance, and to Prof. R. RIEGER for critical reading of the manuscript.

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