Gene,7 (1979) 317--334 317 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

M ORGANIZATION OF A NUCLEAR DNA SEQUENCE FROM A HIGHER PLANT: MOLECULAR CLONING AND CHARACTERIZATION OF SOYBEAN RIBOSOMAL DNA

(Phage), vector; Charon 4A; Glycine max; repetitive DNA; restriction endo- nuclceses; recombinant DNA)

AVIVA VA/~ANYI-BREINER*, JAMES F. GUSELLA, CHERYL KEYS and DAVID E. HOUSMAN

Center for Cancer Research and Department o f Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 (USA)

and

DANIEL SULLIVAN, NORMAND BRISSON and DESH PAL S. VERMA

Deputment o f Biology, MeGiff University, Montreal, Quebec H3A 1BI (Canada)

(Received June 6th, 1979) (Accepted July 21st, 1979)

8 M A R Y

The recombinant DNA vector, ~, Charon 4A, was used to construct a library of DNA sequences from the genomic DNA of soybean (G/ycine max). To define the organization of ribosomal DNA (rDNA) in the soybean genome, clones containing sequences complementary to both 17S and 25S rRNA have been isolated from this library and used in conjunction with Southern blot hybridization. The rRNA genes are tandemly reiterated with a relatively small unit repeat length of 7.8 kb. There is no heterogeneity in the length of the rDNA repeat units although they display limited differences in either base se- quence or pattern of methylation. The cloned rDNA sequences are shown to comprise the entire repeat unit and have been used to obtain a detailed restric- tion map as well as an approximate transcription map of soybean rRNA genes. The cloning of rDNA from soybean suggests that recombinant DNA tech- niques can be successfully applied to the genomic DNA of higher plants despite the high degree of methylation exhibited by plant DNA.

INTRODUCTION

The genes for ribosomal rRNA in most eukaryotes are organized in tandem- ly repeating transcription units (Maizeis, 1976; Wellauer et al., 1976; Amheim

*Present address: Department of Chemical Immunology, We',~.mann Institute, Rehovot (Israel). Abbreviations: kb, kilob~e pairs; 51)8, sodium dodeeyl sulfate.

318

and Southern 1977; Bell et al., 1.977; Cory and Adamg, 1977; Pellegrlni et al. 1977; We!!~;er and Dawid, 1977; Manning et al. 1978; Philippsen et al. 1978a). A great deal of information on the or~jmiT-atlon and ftmction of these genes has been geined by applying molecular cloning methods to ribosomal DNA (rDNA) (McClements et al., 1977; Tiemeier et al., 1977; Dawid et al., 1978; Manning et al., 1978; Philippsen et al., 1978b; Wellsuer and Dawid, 1978; and Gnnnmt et al., 1979). While the genetic organization of the nuclear DNA of higher plants has been an area of great interest (Goldberg, 1978; Rimpan et al., 1978), molecular cloning methods have not been applied to the nuclear DNA of plants. In the present study we have used molecular cloning to study the ribosomal RNA genes of the soybean, Glyeine max. We report here the isolation of several DNA segments which have been cloned using a ~, phage vector. The rDNA is made up of a repeat unit of 7800 base pairs. ThiA unit is shown to include the coding regions for both 17S and 25S rRNA and the organization of the trauscfiption unit has been determined.

MA'I'ERIAI~ AND METHOI~

Preparaffon o f soybean DNA DNA was prepared from soybean (Gly¢ine max vat. prize) embryos which

had been soaked in distilled H20 for 2--4 h. 50 g of embryos were pulverized in liquid nitrogen with a mortar and pestle end then suspended in 150 ml of buffer containing 100 mM NaCI, 50 mM EDTA pH 8.0, 2% 8D8. Prommse (Sigma) (preincubated at 70°C for 5 min in 0.1 X SSC at a concentration of 5.5 mg/ml) was then added to the resuspended embryos at a final concentra. tion of 400 #g/ml. After I h of incubation at 37°C, the slurry was centrifuged at 8000 × g for 10 min at 20°C. The pellet was reextmct~ with 50 ml of 0.1 X SSC and all supematants were pooled. Sodium perchlorate (pH 7.0) was added at 0°C to a final concentration of 0.5 M. After 10 rain at 0°C the prepa- ration was extracted (gentle ah~klng for 20 rain) with an equal volume of phenol (premturated with 0.1 M Tris-HCl pH 7.6 / 0.01% (w/v) 8-hydroxy- quinoline):chloroform:isomnyl alcohol (25:24:1). The emulsion was cleared at 12 000 × g for 10 min. The interface and orgmfic phase were reextmcted with 20 ml 0.1 X SSC end the aqueous frvctious were then pooled and reex- tracted with an equal volume of chloroform:isoamyl alcohol (24:1). DNA was collected by ethanol precipitation and was resuspended in 0.1 X SSC. Pancreatic RNase (preboiled for 5 rain) was added to 20 #g/m! m~d the solu- tion was incubated for 30 rain at 37°C. Pronase (Calbiochem; pretreated as above) was added to 400 #g/ml and incubation was continu¢.<l for 30 min. The solution was extracted with phenohchloroform:isoamyl, alcohol (25:24:1) and the DNA was precipitated with 2 vol. of eth~.~nol. This precipi- tare was redissolved in 0.1 X SSC and the DNA was repell~4~l in a Beckman Type 65 rotor at 50 000 rev./min for 16 h at 4°C. The pe|let was redi~olved in 0.1 × SSC and 1.29 g of solid CsCI was added per ml of solution. This so lu- tion was centrifuged at 35 000 rev./min in a Beckman type 50 rotor at 20°C

319

for 60 h. After fractionatior, of the CsC1 gradient the DNA was located by fluorescence under ultraviolet light after adding 10 ~1 of 50 #g/ml ethidium bromide to 10 #1 aliquots of each fraction. DNA containing fractions were pooled and extensively dialyzed against 0.1 × SSC at 4°C. DNA was then pel- leted at 50 000 rev./min for 16 h (as above), redissolved in 0.1 × SSC and stored at 4°C. The procedure produced yields of 2--3 mg DNA/50 g embryo tissue.

Restriction of DNA Enzymes were purchased from the following suppliers: EcoRI and BamHI,

Boehringer, Mannheim; HindIII and BglII, Bethesda Research Laboratories; XbaI, PouII, KpnI and SacI, New England Biolabs. Digestions were performed at DNA concentrations of 20--100/~g/ml in the reaction mixture specified by the supplier except in the case of EcoRI where digestion was at DNA concen- trations of 20--500 ~g/ml in 100 mM Tris-HCl, pH 7.5, 50 mM NaCI, 10 mM MgCI2, 2 mM ~-mercaptoethanol. Double digests were performed by inacti- vating the first enzyme at 65°C for 5 min, adjusting the concentration of the reaction mix and adding the second enzyme.

Preparation and labeling of soybean rRNA Total polysomes were isolated from 3-week-old root nodules of Glycine

max and were extracted as previously described (Verma et ai., 1974). Poly- somal RNA was separated into oligo(dT) bound [poly(A)* RNA] and unbound [poly(A)- RNA] fractions (Aviv and Leder, 1972). The bound RNA was re- covered by centrifugation at 50 000 rev./min for 16 h at 4°C. Separated 17S and 258 rRNAs were prepared from the total poly(A)- RNA by sedimenta- tion through 10--30% (w/v) sucrose gradients (in 100 mM NaCI, 10 mM Tris.HCl pH 7.5, I mM EDTA, 0.1% SDS) for 11 h at 32 000 rev./min in a Beckman SW41 rotor at 21°C. Appropriate fractions were pooled and the RNA was collected by ethanol precipitation.

Either total poly(A)- RNA or separated 17S or 25S rRNA were end-labeled as follows. 10 #g of the appropriate RNA was lyophilized and resuspended in 4.5 #I of 0.1 M Tris.HCl (pH 8.0). 0.5 #l of bacterial alkaline phosphatase (2.5 units/ml) was added and the reaction mix was incubated for 30 min at 55°C. 0.5 #1 of 50 mM nitriloacetic acid was then added. After 20 min at room temperature, the reaction mix was heated to 100 °C for 90 sec. The entire reaction mix was then added to a tube in which 200/ACi of [7-32P] ATP (3000--6000 Ci/mmol, New England Nuclear) had previously been dried down under a stream of nitrogen. 2/~1 of 75 mM MgCI2, 75 mM O.mercaptoethanol were sdded along with 2 ~1 H20. The reaction was initiated by addition of 2 units of polynucleotide kinase (Boehringer, Mannheim), continued for 30 rain at 37°C, and terminated by heating to 100°C for 60 sec. Labeled RNA was separated from triphosphate on a Sephadex G-150 column buffered with 10 mM Tris.HCl (pH 7.9), 100 mM NaCI, I mM EDTA.

320

Pre~mtion of phoge DNA ACharon 4A was propagated essentially as described by Blattner et ai.,

(1977) with the exception ~ E. coil LE392 (Tiemeier et al., 1978) was used as host. DNA was prepared from 500 ml phage lysates as follows. 30 g of NaCI was added to the lysed culture snd then bacterial debris was pelleted at 8000 rev.~in for 10 mln ~Sorvall GSA rotor). 35 g polyethylene glycol 6000 (Sigma) was added to the supematant and di~olved by stirring at room tem- p . Stirring was then contin-ed at 4°C for a minimum of I h. The PEG precipitate was collected at 10 000 t~v./min for 20 min and redissolved in a final volume of 10 m! buffer (10 mM Tris-HCl pH 8,0,100 mM NaCI, 10 mM MgCI~). 7.~ g solid CsCI was added and the phage was banded at 45 000 rev./ mln in a Beckm~m Type 65 rotor for 24 h at 20°C. The phage band was col-

through the side of the tube with a syringe and then rebanded under the same conditions as above. The phage was then dialyzed extensively against buffer P at 4°C. The dialyzed phage solution was adjusted to 0.3 M NaCl and extracted twice with phenol and 3 times with ehlomform:imamyl alcohol (24:1). DNA was collected by ethanol precipitation and stored at 4°C in 10 mM Tris pH 7.9, I mM EDTA. Yields of Charon 4A vector And recom- binant clone DNAs were 0.2--1.5 mg/liter of culture.

Construction of soybean library in ACharon 4A 2.5 ~g £coRI cut soybean DNA (average fragment size § kb) was mixed

with 3 #g EeoRI cut Charon 4A DNA in a volume of 18 ~1 of 100 mM Tris. HCI pH 7.5, 50 mM NaCI, 10 mM MgCI2, 2 mM j3.mercaptoethanol. 1 ~1 of 20 X ligase salts (5 mM ATP, 0.5 M MgCI2,1 M dithiothreitol) and I pl of DNA ligase (Miles, 400 units/ml)were added and incubation carded out at 4°C for 48 h. Ligase was inactivated at 65°C for 5 min and the DNA was packaged into intact phage particles by the in vitro encapsidation procedure described by Blattner et al., 1978.

Phage were plated after a 10-min preadsorption (37°C) in a mixture of 0.1 ml saturated bacteria (LE392) and 0.1 ml adsorption solution (10 mM MgC12, 10 mM CaCI=). When dilution was required, phage were diluted in ad- sorption solution. NZY broth (Blattner et al., 19",7) was used for maintaining L~.392 and for preparing phage lysates. NZY + 1.1% agar was used as bottom agar and NZY + 0.65% agarose used as top agar for plaquing. The in vitro en- capsidation procedure yielded a total of 2.6- 104 pfu of phage.

Plate stocks of this soybean library were prepared by scraping the top agar containing phage plaques and pelleting the agar and bacterial debris at 10 000 rev./min for 10 rain. The supematant liquid was diluted into 10 mM Tris (pH 7.5), 100 mM NaCI, 10 mM MgCI2, 0.1% gelatin and stored at 40C over chloroform.

Screening of soy bean library for rDNA-containing recombinants Phage from the soybean library were plated at 1000 plaques/plate as de-

scribed above. Nitrocellulose filters were prepared for each plate as described

321

by Benton and Davis (1977). Hybridization was performed as described by Malfiatis et al. (1978) using 5 - 10 s cpm/filter s2P-poly(A)- soybean RNA. Plaques hybridizing to the probe were detected by autoradiography for 24 h at --70°C using Ko0~k XR-5 X-ray film in conjunction with an intensifying screen (Dupont Cronex). These plaques were twice replated and rescreened as above to insure their purity. The rec0mbinants were then propagated on LR392 according to Blattner et al. (1977).

All procedures involving the construction of the soybean library and sub- sequent screening and amplification of recombinants were performed at the P2-EK1 level of containment as required by the NIH Guidelines.

Agarose gel electrophoresis and transfer of DNA to nitrocellulose ~ c t e d samples of DNA were adjusted to the same salt concentration

and 40% Ficoll (containing bromophenol blue as dye marker) was added to a final concentration of 10%. Electrophoresis of DNA was at 3 0 - 4 5 V for 12--20 h on a 20 cm vertical agarose gel apparatus (Blaircraft Inc.) in E buf- far (40 mM Tris.HCI[pH 7.6], 20 mM sodium acetate, 1 mM EDTA). Unless otherwise specified, a XeI8578am7 HindIH digest was used to provide size markers.

DNA was vi~mliT~ffid trader ultl~/Lviolet light after staining for 30 min with 0.5 pg/m! ethidium bromide. The gel was then treated with 1 N NaOH for 45 rain, 1 M Tris pH 7.6, 1.5 M NaCI for 45 min and DNA transferred to nitrocellulose as described by Southern (1975).

Total poly(A)- RNA was run on a 1% agarose gel in the presence of methylmezcuric hydroxide and transferred to diazotized cellulose as de- ~ribed by Alwine et al. (1977).

Preparation of nick.translated DNA Because the rDNA clones contained some soybean EcoRI fragments not

hybridizing to rRNA (data not shown) it was necessary that rDNA be isolated from these clones to be used as a hybridization probe, rDNA was isolated from £coRI<tigested clone Gin2, Gin9 or Gm3 by agarose gel electrophoresis fol- low~i by freeze4queezing the appropriate restriction fragment from the gel (Th,~ng et al., 1975). This DNA was then labeled to high specific activity (5.10~--10 s cpm/,g) by the nick translation method of Rigby et al. (1977) and used as probe against filters continuing soybean DNA or RNA. This method resulted in rDNA probe which was only slightly contaminated with other soybean sequences. These contaminants occasionally produced very faint bands on autorediograms which were easily distinguished from rDNA mm~ction fragments.

When the isolated rDNA was to be used for mapping experiments, it was nick translated to lower specific activity (2--5- l 0 s cpm/pg). This DNA could then be digested with various restriction enzymes, and run on an agarose gel. When the gel was dried and autoradiographed, the fragment sizes could be determined with accuracy using ~X174 markers prepared in a similar f~hion.

llyb "ndization of f~ters Hybridization of filters containing bound DNA or RNA was performed as

described by Maniatis et al. (1978) for Benton-Davis filters. Autoradiography was at--TG°C for 1--7 days rating Kodak XR-5 film and an mtendfying screen (Dupont Cronex).

RESULTS

PreUminm~ mapping of soybean rDNA A prelimbmry analysis of the structure of the rDNA of Glyclne max was

performed by Southern hylzdd!-~on of [mP]rRNA to Glycine max nuclear

-X

m x a}

-fj ~ o

4.1-- . '" ':'";:

- e _ , - - 5 - ~ -

(O UJ %~f~* * ? , , , _

;49

Fig. 1. Hybridization of soybean rRNA to restriction nuclease digested soybean DNA. Soybean DNA digested with the indicated e n z y m u was subjected to agarose gel eleetm- phoresis in a 1.4% agarose gel and transferred to nitrocellulose as described in MATERIA/~ AND METHODS. The nitrocellulose f'dter was hybridized to 5" 10 s epm of 3~P4abeled soybean rRNA (S.A. 5" 10" cpm/~g) and the pattern of hybridization determined by autoradiography. I pg of restriction nuclease digested soybean DNA was used per lane. Fragment sizes were determined using ~ HindlH markers run in parallel.

323

DNA digested with a number of restriction enzymes. These experiments led to the conclusion that Glycine max appears to have a single major rDNA re- peat of approx. 8 kb. ZYperiments consistent with thig conclusion are shown in Fig. 1. ht Fig. l a soybean nuclear DNA digested with BglH, XbaI and BglH + XbaI has been fractionated on a 1.4% agarose gel and hybridized to 32P-labeled rRNA. XbaI and BgllI each exhibit a single major band at 7.8 kb. Digestion in the case of XbaI is complete; no hybridization is observed out- side the 8 kb region. Digestion with BglIl is incomplete. Hybridization to the probe is observed in the region of the gel in which undigested DNA migrates. These results are consistent with a single 7.8 kb repeat unit for G/ycine max rDNA containing a single XbaI site and a single BglIl site. Con- trol experiments in which ), DNA was mixed with soybean DNA demonstrate that the incomplete digestion of soybean rDNA with BglII must be due to the absence of BglH sites or to methylation of these sites in some soybean rDNA repeats (data not shown).

Digestion with 8acI (Fig. 1B) yields three DNA fragments 1.7 kb, 2.3 kb and 3.9 kb which hybzidize to 3ZP-labeled rRNA. Digestion with EcoRI yields two DNA fragments of 3.8 kb and 4 kb which are clearly resolved on 1.4% agar0se gel (Fig. 1C). The fact that 8acI and EcoRI yield hybridizable f~gments which sum to a total of 7.8 kb is also consistent with a 7.8 kb re- peat length for the rDNA of Gly¢ine max. Further results consistent with this conclusion are the results of an Xbal + BglII double digest (Fig. 1A) which yields three hybridizable fragments of 3.7 kb, 4.1 kb and 7.8 kb. This pattern is consistent with a 7.8 kb repeat length in which all copies are sus. eeptibie to XbaI digestion but only a portion of the copies ate susceptible to Bglll. Digestion with £coRI + Xbal (Fig. lc) yields three fragments of 1.4 kb, 2.6 kb and 3.8 kb. This result is comistent with the presence of two EcoRI sites and one XbaI sites per 7.8 kb repeat, Digestion with £coRI and 8acI (Fig. lc) also yields three prominent bands of 1.6 kb, 2 kb and 3.7 kb. Based upon these results a preliminary map shown in Fig. 2 was constructed.

Xbo[ 8glU Xbol BgtH . . . . ~ ~ ~ ~ . . . . . .

f t ! t t EcoN EcoRI EcoRI EcoR/ EcoRl

3JlO 4.0 EcoRI I, I ,

7.8 Sgt[ , ,

7.8 Xbe l I

3.7 4.1 Bg|X +Xbo [ I I I

Fig. 2. A preliminary map o f restriction enzyme sites in the soybean rRNA repeat. Place- ment of restriction sites is based on data shown in Fig. 1.

324

l~olation and characterization of XCharon 4A donas carrying a soybean rDNA ~n~mrt

To fm'ther analyze the OXlPmizafion of the r.DNA, a library of DNA clones of Glye/ne max DNA was established using ~,Chazon 4A (Blather et ai., 1977) as a vector. Clones eazzying r.DNA sequences were seleetedfzom the librazy by hytn'idiufion of [~P]rRNA to DNA of plaques from the libm.w using the plaque hyb "ndisafion proeedure of Benton and Davis (1977). Approximately one in 500 of the plaques in the library exhibited hybridization to the [ ~ ] . rRNA probe. Nine clones were selected for initial chm'sctaiza~ou. DNA from each clone was ~ with EcoRl done, £eoRl + Xbal and £eoRl + Bglll, haetionated on a 1.4% a p m s e gel and hybridized to [~P] rRNA. Three basic hy "tmdizafion pagtems were obsegved. Clones Gin8 and Gin9 appeEed to in- dude an EeoRI fawgment of 4 kb which could be digested completely with

EcoR! • ECOR!+Xba! r - • • ' t

A O C D A B C D

Fig. 3. Hybridization of soybean rRNA to DNA of clones Gin2, Gin3 and Gm9. 0.1 ~g of purified DNA of clones Ore3, Gin9 and (}m2 and 1 0zg of soybean DNA were dipsted either with EcoRI or with X~s[ followed by EeoRl. The digested DNA was fraetionated on a 1.4% qarose gel, tmnsfenmd to a nitrocellulose lifter and hybridized to ~ P soybean rRNA as described in Fig. 1. and MATERIALS AND METHODS. (A) ChaA-GmS; (B) Ch4A.Cdng; (C) Ch4A-Gm2; {D) mybean pnomie DNA.

325

XbaI but which was insensitive to BgIH digestion. Clones Gin1, 2, 4, 5, 6 and 7 included an EcoRI fragment o f 3 .8 kb which digested with BgIIi but which was ingensitive to the XbaI digestion. Clone Gin3 contains t w o EcoRI frag- ments, which hybridize to rRNA, one 3 .8 kb and one 4.0 kb. The 4 kb frag- ment produced by EcoRI digestion of GM3 was sensitive to digestion by XbaI and insensitive to BgllI while the 3.8 kb fragment was sensitive to BglII and insensitive to XbaI digestion. The hybridization pattern of a single clone representative o f each group is shown in Fig. 3.

_ - . _ , _ I I ' I V i " - - I " E

;" ~" ~:;,~ . ~ ; ..... 2.6

i

Fig. 4. Hybridization of clones Gm2, Gin3 and Gm9 to restriction nucleue digested soy- bean DNA. 1 ~ of soybean DNA digested with either EeoRl alone, Xbal alone or Xbal fol- lowed by EcoEl was subjected to eleetrophoresis on a 1.4% a~rose gel and transferred to a nitro~lluloN filter. ~,parate rdtem were hybridized to a [s*P]DNA probe (5-10 ~ cpm/~) pmpmd from the purified soybean rDNA sequences of clones (Ires (A), Gm2 (B) and Gin9 (C) res11~etlvaly. PurlFmation of the rDNA Nquenees from the recombinant clones and nick tr~ lat ion to produce a 3:p probe was performed as described in MATERIALS

METHODS.

326

The hybridization patterns of all the clones we have isolated are consistent with the t e n _~iv_" e map in Fig. 2. We chose one member e~hibiting each pat- tern of hybridization for further study.

To analyze the homology between clones Gin2, Gm3 and Gm9 with gen- omic DNA we performed a series of experiments in which cloned DNA was used as a probe in a 8outhemhybridization against nuclear DNA disested with EcoRI, XbaI and EcoRI + XbaI. As shown in Fig. 4, clone Gin2 hy- bridizes exclusively to a 7.9 kb fn~ment produced by XbaI cleavage to a 3.8 kb fragment produced by EcoRI digestion and to a 3.8 kb fragment pro- duced by the comb;reed EcoRI and Xbal digest. Tiffs result is consistent with the map shown in Fig, 2 and the assignment of clone Gin2 to the left hand £coRl frasment on that map. Clone Gin9 hybridizes to a 7.9 kb Xbal frag- ment and 4.0 kb EcoRl fragment. 3~P-labeled Gin9 DNA hybridizes to two DNA fragments in the EcoRl and Xbal double digest of 1A kb and 2.6 kb. No hybridization is observed at 4 kb with Gin9 probe and this doubly digested DNA. These results are consistent with the assignment of Gin9 to the right hand £coRl fragment on the m~p in Fig. 2. Clone Gm3 shows hybridiza- tion to a 7.9 kb fragment produced by Xbal digestion, a pa/r of closely spaced fragments produced by EeoRI digestion of about 3.8 kb and 4 kb and three fragments of 1A kb, 2.6 kb and 3.8 kb produced by combined £coRI and Xbal digestion.

These results suggest that the Gin3 clone includes the entire rDNA repeat. This clone is likely to be the product of incomplete £coRl digestion since the probability of clones carrying such fragments by chance is about 1/500.

Further analysfs of the organization of soybean rDNA The results obtained thus far do not completely rule out the potential pre-

sence of a nontranscribed spacer sequence adjacent to the coding unit. To determine whether Gin2 and Gin9 together include the entire 7.8 kb repeat, each clone was hybridized to a double digest of genomic DNA with BgiIl and Xbal. If the entire rDNA repeat length is significantly greater than 7.8 kb then these two clones should hybridi,~ to different sized fragments in a Bg/II + XbaI digest. In fact the same basic pattern of hybridization is ob- served (Fig. 5) when DNA of each clone is used as a hybridization probe against the double digest of genomic DNA. This result is consistent with the map shown in Fig. 2, but is inconsistent with the presence of a non-transcribed spacer sequence adjacent to the 7.8 kb repeat unit.

Further evidence on this point was obtained by performing a series of diges- tions of soybean DNA with decreasing nmounts of £CORL At the lower EcoRI concentrations partial digestion of the rDNA segments has been achieved. If the map of the rDNA shown in Fig. 2 is correct then hybridiza- tion of Gm2 probe to partially digested rDNA should be observed only to DNA fimgments of 3.8 kb, 7.8 kb and 11.6 kb, 15.6 kb, etc. No additional class of fragments hybridizing to the probe should be observed in the partial EcoRI digest. This result was obtained and is shown in Fig. 6.

327

decreosing EcoR[ Xbol

A

kb i - - - - 7 . 8

B

P

kb . . . . ~ - 2 3

~ ~:~ - " -9.8

-4 .5

-2 .5

-2 .2

Fig. 5. Hybridization of DNA of clones Gm2 and Gm9 to soybean DNA digested with Xbal + Bg/II. Procedures were similar to Fig. 4 except that digestion of soybean DNA was performed with restriction enzyme BglII followed by XbaI. Probe: (A) Ch4A'Gm2; (B) Ch4A-Gm9.

Fig. 6. Hybridization of [3=P]rDNA clone Gm2 to soybean DNA partially digested with EcoRI. 1 #g of soybean DNA was digested with Xbol or with lX, 1/4×, 1/8× the amount of EcoRI required for complete digestion and was fractionated on an 0.7% agarose gel. The DNA was transferred to nitrocellulose and hybridized to nick-translated [32P]rDNA (3.8 kb fragment) isolated from done Gm2 as described in MATERIALS AND METHODS. The positions of x HindIII marker fragments run in a parallel lane are indicated.

$28

To ~ map the clones the DNA fragments ~ rDNA from Gm2, Gm9 and Gm3 were isolated, labeled with ~zp by nick translation (Rigby et aL, 1977) and digested with a series o f r ~ n enzymes. The results o f one such experiment are shown in Fig. 7 whUe the results of a series of these ex- pmiments are tabtdat~ in Table I. Based on these results a det.sfled map of the ~ n enzyme sites present in the soybems rDNA repeat was con- stmcted (Fig. 10).

Fig. 7. Agarc~ gel e lec t ropho~is of rest~lotion enzyme digested soybems rDN& from clone Gin3. Cloned soybean rDNA was prepared from EcoRl digested DNA of clone GmS. This DNA was labeled with 3~p by the nick trans!al tion reaction and further digested with the indicated restriction enzymes. The digested DNA was fractionated on a 2% nfJnrose gel. Fragments of ~X174 DNA similarly 3~P-labeled and digested with restriction enzymes Pstl, Haelll and Taql, were used as precise size msrker~ Lanes: (1) EcoRl; ( 2 ) / ~ m H l ; (8) sacI; (4) xbaI; (5) ~ m ; (6) gpn[; (V) ~

329

TABLE I

R ~ T R I C T I O N FRAGMENTS OF CLONED zDNA

Data weze derived f t~m Fig. 7 and .im.,,s- experiments using Ch4A • Gin2 and Ch4A - Gm9 rDNA inserts. N.D.. no t done

INgest/on Fzsgment s/zes (kb)

Ch4A'Gm2 Ch4A'Gm9 Ch4A'Gm3

EcoRI 3.80 4.00 4.00, 3.80 BamHl 1.60, 1.18, 0.98 1.68, 1.21, 1.03 1.67, 1.60, 1.19, 1.04, 0.98 8acl 2.05, 1.60, 0.20 3.70, 0.35 3.65, 2.03, 1.60, 0 .34 .0 .20 Xbal 3.90 2.63, 1.42 3.80, 2.65, 1.40 Bl | 2.81. 1.80 4.00 4.00, 2.33, 1.48 Kpnl N.D. N.D. 4.00, 3.85 Pvull N.D. N.D. 4.00, 3.85 Hindfl l 3.35 4.05 4.00, 3.80 BamHl + Bglll 1J~50 1.03, 0.73, 0.49 N.D. N.D. B#lfl + 8ael 2.05, 1.23, 0.35, 0.20 N.D. N.D. BamHl + 8 ~ I 1.60, 0.85, 0.78, 0.38, 0.20 1.70, 1.15, 0.75, 0.34 N.D. BamHl + Xbal N.D. 1 .70,1.10, 0 ~ 6 , 0.35 N.D. Sacl + Xbal N.D. 2.61, 1.0,5, 0.85 N.D.

Transcriptional mapping of soybean rDNA A further point of interest is the direction of transcription of the rRNA

genes and the identification of which DNA segments code for each rRNA species. An initial experiment to determine whether Gm2 or Gin9 completely encodes either the 178 or 258 RNA genes was designed as follows. Glycine max rRNA was fractionated by agarose gel electrophoresis and bound to DBM.cellulobe by the procedure of Alwine et al. (1977). The filter-bound RNA was hybridized separately to 32P-Gm2 DNA and 3~P-Gm9 DNA. Clone Gm2 DNA and clone Gin9 DNA each hybridized to both 178 and 25S sequences (data not shown). These results indicate that each clone codes for a portion of each rRNA.

To clarify the relationship between the cloned DNA segments and each rRNA a series of experiments in which 17S and 25S RNA were hybridized separately to DNA fragments produced by restriction digestion of clone Gm3 with EcoRI, SacI + £coRI and XbaI + EcoRI. Results are shown in Fig. 8. These results allow an approximate assignment of the DNA segments which code for 178 and 258 rRNAs but do not permit determination of the direc- tion of transcription. To make this determination we have prepared a probe which corresponds to only the 5' end of each molecule by end-labeling intact rRNA with polynucleotide kinase followed by partial alkaline hydrolysis of the RNA probe prior to hybridization. These probes were then hybridized to appropriate restriction enzyme digests of rDNA isolated from Gm3. The re- sults of this hybridization are shown in Fig. 9. In each lane a single DNA frag- ment hybridizes most intensely to the 5' end specific probe. Using these re- sults an assignment of the position for the 5' end of the 17S rRNA and 25S rRNA has been made. These assignments are shown in Fig. 10.

SSO

25S Probe A B C

W

17S Probe A B C

25S 5' probe A B C D

17S 5 '- probe

A B C D

2 -

Fig. 8. Hybridization o f restriction nuclease digested DNA of clone Gm3 to separated 17S and 258 rRNA. 0.3 ~g o f DNA o f alone Gin3 was digested with the indicated restriction enzyme or enzymes. The digested DNA was subjected to gel electrophoresis on a 1.4% agerose gel and transferred to a nitrocellulose fRter. 17S and 258 rRNAs were fractionated as described in MATERIALS P ND METHODS and used separately as a hybridization probe (2 • 10' cpm ==P/l=g RNA)to the restriction nuclease digested DNA of clone Gin3. Lane: (A) ~ I + EeoRI; (B) XbaI + EcoRI; (C) EcoRI.

Fig. 9. Hybridization o f restriction nuclease digested DNA o f clone Gin8 to 5"-specific 17S and 25$ rRNA probes. 0.3 #g o f DNA of clone Gm3 was digested with the indicated restrietion enzyme or combination o f enzymes. The digested DNA was subjected to gel electrophoresis on a 1.4% agerose gel and transferred to a nitrocellulose filter. 17S and 258 rRNAs were fractionated as described in MATERIAI~ AND METHODS. Each was then end4abeled using [~2P]ATP and subjected to alkaline degradation in 0.3 N NaOH at 37°C for 40 min. The reaction was neutralized using an equal volume of 0.3 N HCI and the partially degraded rRNAs were used as hybridization probes against separate filters as described in MATERIALS AND METHODS. Lane: (A) BglII + E¢oRI; (B) Sacl + E¢oRI; (C) XbaI + EcoRI; (D) EcoRI.

l_ Repeat Unit of Ribosomal DNA • i -C~

17S F_. 25S ~ .¥ 17S ~' 5S - ~ r-- - -_-::':_~ rRNA I

~i i x s ~i i i i i e "0 [ i ' i L s' ' [ -'rDNA

I.

I

I A £ - EeoI~

J • O • Ogmlfl

i ~ Og- s i gn i o S • Sacl l e X • Xlml. | ? ! z,,b

Ch4A.Gm3 7.8 kb

Ch4A-Gm2 3.8 kb I

Ch4A. Gm 9 ! [ 4£) kb

Fig. 10. A detailed map of restriction enzyme sites and transcriptional organization of soybean rDNA. The data compiled in previous figures and tables have been used to develop the composite map of soybean rDNA. PvulI, KpnI and HindIH are non-cutters for the rDNA repeat unit. The 3.8 kb and 4.0 kb rDNA inserts of Ch4A'Gm3 were aligned with respect to each other using digestion with SacI and with digestion with Bg/H + Xbal (data not shown).

331

DISCU88ION

The soybean genome has a kinetic complexity of 1.8.10 e kb (Goldberg, 1978). Approx. 1400 copies of rDNA are present in this genome (K. Lark, personal communication). The frequency of rDNA clones in the library used in this study is within the range expected if our library contains a representa- tive selection of soybean nuclear DNA sequences.

A number of specific features of soybean rDNA sequences deserve comment. (I) Length heterogeneity. A number of eukaryotes including Drosophila

melanogaster (Wellaner and Dawid, 1977), Xenopus laevis (Wellauer et al., 1978; Botchan et al., 1977) and man (Krystal and Arnheim, 1978) exhibit significant heterogeneity in the length of the ribosomal DNA repeat unit. The silkworm, Bombyx mori, however, exhibits no detectable heterogeneity in the repeat length of the rDNA (Manning et al., 1978). The analysis of soybean rDNA suggests a pattern similar to Bombyx mori in that no significant length heterogeneity in the soybean rDNA repeat unit is observed. This lack of vari- ability in repeat length is a genetic characteristic which may prove of interest in analyzing the functional characteristics of rDNA.

(2) Length of possible spacer DNA sequences. Soybean rDNA has a repeat length of 7.8 kb. Approx. 2 kb is xequired to code for 17S rRNA and 4 kb is required to code for 25S rRNA. A 42S precun~r to plant rRNA has previous- ly been reported (Cox and Turnock, 1973). Such a precursor would require approx. 7 kb of DNA coding capacity. Clea~ly the amount of rDNA present as non-transcribed spacer in soybean rDNA cannot be significantly greater

332

thnn I kb. A further question of interest is whether the coding sequence for any additional RNAs such as 5S RNA are contained within the 7.8 kb re- peat unit. The relatively small length of spacer DNA sequences in the soybean rDNA is similar to the short spacer length of Bombyx mori (Mmming et al., 1978) andyeast rDNA (Philippsen et al., 1978a) and nnllke the comparatively long spacer in Mus muscuhts (Arnheim and Southern, 1977; Cory and Adams, 1977).

(3) Presence of possible sequence heterogeneity in the rDNA repeat. The pattern of digestion with a number of restriction nuclesses of cloned soybean rDNA and rDNA isolated directly from soybean cells are in most cases iden- tical. In at least one case (Fig. 1), digestion with BglH, a ~4gnificant d;~ference is observed between cloned DNA and soybean nuclear DNA. In seven indepen- dently isolated clones the BgHI site shown in Figs. 2 and 9 is present in the cloned rDNA segment. Digestion of soybean nuclear DNA with Bg/ll strongly indicates that some copies of the soybean rDNA repe~t are susceptible to Bgfll digestion and others are not. Analysis of a combined BgIH--Xbal r~Ms- tion suggests that at least half the rDNA repeats cannot he digested with /~/II. This result could either indicate that the BgIH recognition sequence, AGATCT, is missing from these copies of the rDNA repeat or could he due to methylation of the BglII DNA recognition site in these copies. The fact that all of the 7 clones tested have a BgHI recognition site suggests either that copies of rDNA containing the BglH ~,e are incorporated preferentially into recombinant DNA molecules or that the methylation explanation is correct. The latter possibility is strengthened by the high degree of methylation of the rDNA which we have observed at certain other restriction nuclease recogni- tion sites (Varsanyi.Breiner, et al., unpublished observations).

(4) Organ~ction of rRNA transcription. The results presented here are compatible with transcription of the rRNA sequences without interruption. However, the presence of an intervening sequence of approx. 500 base pairs or less in the 178 or 258 rRNA genes cannot he ruled out based on the pre- sent data. The occurrence of an intervening sequence in rDNA has been re- ported in Tetrahymena p~/riformis where it is transcribed and spliced out (Wild and Gall, 1979), and in Drosophila mebmogaster where rDNA repeat units carrying the inserted sequence may be transcriptionally inactive (White and Hogness, 1977).

The ability to produce a library from soybean nuclear DNA opens the prospect of a detailed analysis of the gene organization of higher plants using recombinant DNA techniques. Some question as to the feasibility of apply- ing recombinant DNA techniques has m due to the high degree of methylation of plant DNAs (Shapiro, 1976). It is useful to note in this con- text that the soybean ribosomal DNA appears to he highly methylated. De- spite this fact, it .w~ cloned in Charon 4A with a high efficiency. It should also he pointed out that the library of soybean DNA clones reported here clearly contains a large number of other soybean DNA sequences. Further analysis of the soybean genome can now be carried out using specific probes such as leghemoglobin eDNA (Baulcomhe and Verma, 1978).

333

ACKNOWLEDGEMENTS

This research project was suppor ted by a grant from the Nat iona l Science Founda t ion (No. 76-17747) to D.H. and b y grants f rom Natura l Sciences and Engineering Research Counci l of Canada and Rockefel ler Founda t i on to D ~ . S . V . N . B . is t hankhf l for a graduate fel lowship from N.S.E.R.C. Canada. The au thors are indebted to Virginia Weeks, Dr. P. Bresmer and Dr. V. Vol loch for runn ing me thy imercu ry gels, and Dr. B. Goodchi ld for reading the manu- script.

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Communicated by A~I. Skalka.