CELL STRUCTURE AND FUNCTION 17: 87-92 (1992)

© 1992 by Japan Society for Cell Biology

Isolation and Characterization of Nuclei from Rice Embryos

Junji Yamaguchi, Poh-Yam Lim, Kazumi Aratani1) and Takashi Akazawa*Research Institute for Biochemical Regulation, School of Agricultural Sciences, Nagoya University, Chikusa,Nagoya 464-01, and ^Ichimura Gqkuen Junior College, Uchikubo, Inuyama 484, Japan

Key words: histone/nuclear protein/nuclei/rice embryo/RNAsynthesis

ABSTRACT.A method has been developed to isolate pure preparations of nuclei in high yield from commer-cially available viable rice embryos (germ), employing extraction with buffer solution containing glycerol(without detergent) and polyamine, followed by centrifugation on a 30% Percoll cushion. The intactness of theisolated nuclei was confirmed by light microscopy as well as electron microscopy. The protein profiles of both

whole nuclei and nuclear extracts obtained by SDS-PAGE,organellar marker enzymeactivities, DNAand RNAanalyses, and in vitro RNAsynthesis, all indicate that the highly purified nuclei are isolated from rice embryos.

The biosynthesis of proteins encoded by the nucleargenomeconsists of sequentially oriented networks com-partmentalized in the cell; DNAtranscription and RNAprocessing occur in the nuclei and subsequent RNAtransport and translation of mRNAin the cytoplasm.Although the detailed mechanisms of each of thesesteps has been the subject of intensive cellular and mo-lecular biological studies, RNAsynthesis in the nuclei isof particular interest and importance because of therole of proteins specifically involved in the transcription-al regulation. In recent years muchresearch interest hasbeen focused on elucidating the mechanismsof tran-scriptional regulation using isolated nuclei of plant ori-gin (2, 8, 17). One can thus readily recognize that isola-tion of intact nuclei is of prime importance to study theimport mechanismacross the nuclear envelopes of vari-ous regulatory proteins engaged in the transcriptionalactivities of the nuclear genome. Since signal se-quence(s) of amino acids relating to the import of nu-clear proteins have been characterized in the mamma-lian system (4, 18), it is desirable to construct a feasibleexperimental system using plant materials to advanceour knowledge concerning the mechanism(s) operating* To whomcorrespondence should be sent.

This research was supported in part by Grants-in-Aid-No.02453124 (T.A.), No. 02760049 and No. 02242104-for Special Re-

search on Priority Areas (Cellular and molecular basis for reproduc-tive processes in plants) (J.Y.) from the Ministry of Education, Sci-ence and Culture (Monbusho).This is paper No. 24 in the series "Enzymemechanism of starchbreakdownin germinating rice seeds".Abbreviations used: ADH,alcohol dehydrogenase; DAPI, 4',6-diaminino-2-phenylindole-dihydrochloride; G6PDH, glucose-6-P de-hydrogenase; HMG, high mobility group; NGM, nuclei-grinding me-dium; 6PGDH, gluconate-6-P dehydrogenase.

in their gene expression.It is knownthat the nuclear pore complex is closely re-lated to protein import into the nuclear matrix (6, 18,22). As with other organelles, the isolation of intact nu-clei requires the preservation of the integrity of the nu-clear envelopes. In general the use of non-ionic deter-gents such as Triton X-100 causes the deterioration ofthe nuclear envelope membranes. Another serious prob-lem inherent to the isolation of nuclei from plant tissuesis the inevitable use of physical shear to break up thecell wall. Furthermore, special caution must be given tosuppress the DNase, RNase and protease activitieswhich can readily degrade the nuclear componentsduring isolation (10, 29).We have recently been engaged in a study exploringthe mechanism(s) of transcriptional regulation of a-amylase gene during the germination of rice seeds (1,19, 20), and toward this end we are attempting to char-acterize the regulatory protein components. The isola-tion and characterization of pure nuclei from rice em-bryos, which could help us to achieve this goal, con-stitute the work described in this communication.

MATERIALS AND METHODS

Rice embryos. Embryotissues of rice {Oryza sativa var.Nipponbare) used in this investigation, which was kindly pro-vided to us from Gekkeikan Shuzo Co. Ltd (Kyoto), wasstored at 4°C prior to use. Alternatively, rice embryos wereprepared from fresh whole rice (Nipponbare) seeds by briefhomogenization in a Waring blender. The ground materialwas then passed through a graded wire mesh screen to removeendosperm tissues, and the sieved embryos were floated on amixture of carbon tetrachloride and cyclohexane (25: 10 v/v)

87

J. Yamaguchi et al.

(14). Embryos obtained by either way were viable (more than90%) as determined by ordinary seed germination count.Two-daygerminated rice embryoswere prepared for incuba-tion in 10mM sodium acetate (pH5.2) containing 2mMCaCl2 at 30°C after sterilization (1% NaCIO for lO min).

Isolation of nuclei. Eighty g of dry or germinated rice em-bryos were homogenized for 60 sec and subsequently for 30sec using a Polytron (Brinkmann; PT20ST) in 320 ml of nu-clei-grinding medium (NGM) consisting of 20 mMTris-HCl(pH 7.4), 2mMEDTA, 0.5 mMEGTA, 0.5 mMspermidine,0.15mM spermine, 10mM 2-mercaptoethanol, and 40%

(v/v) glycerol. The homogenate was filtered through 4 layersof cheesecloth and 1 layer of Miracloth (Calbiochem) and 40ml of the filtrate were layered over 10 ml of 30% Percoll (Phar-macia) in NGMin Falcon tubes. The material was centrifugedat 3,500 rpm for 20 min in a swing rotor (Kubota KN-70). Thefraction banding above the 30%Percoll cushion was re-covered and 2 vol of NGMwere added to dilute Percoll. Afterrepeated centrifugation (3,500 rpm, 20 min), the pellet wassuspended in 10ml of NGMand layered over 10ml of 30%Percoll in NGMin an SW-27 tube, and centrifuged for 30 minat 6,800 rpm in a BeckmanSW-27rotor. Nuclei recovered in aband above the 30% Percoll layer was diluted with 2 volumesofNGMand centrifuged at 3,500 rpm for 15 min in a Kubotaswing rotor (KN-70). The nuclei pellet was suspended in 15 mlof NGMand centrifuged again to remove the residual Percoll.The final nuclear pellet was suspended in 1 ml of NGMcon-taining 50% glycerol and stored at -80°C until use.Light and electron microscopy. A small amount of nu-clear preparation was mixed with a drop of staining solutionof 1 //g/ml DAPI in NGMon a slide glass, and observed withan OlympusBHS-RFKepi-fluorescence microscope equippedwith phase contrast objectives, UVFx40PL (Olympus OpticalCo. Ltd., Tokyo, Japan). DAPI was excited by light from a100 Wmercury lamp, using a dichroic mirror and filters forUVirradiation. Photographs were taken with Fujichrome 35mmcolor film of ASA100.For electron microscopy, samples were fixed with 2%(w/v)glutaraldehyde in NGM(containing 50% glycerol) for 2 hr atroom temperature, washed with NGMand then post fixedwith 1% OsO4 for 12hr at 4°C. They were dehydrated in agraded series of ethanol and propylene oxide and embeddedin Epon 812 resin. The this sections were stained with uranylacetate for 1 hr and poststained with lead citrate for 5 min.Enzyme assays and others. Assay methods for marker en-zyme activities and analytical methods are as follows (7):ADH, EC 1.1.1.1. (30): catalase, EC 1.ll.1.6 (15); Cyt cre-ductase, EC 1.6.2.4 (13); DNA (5); fumarase, EC 4.2.1.2 (12);G6PDH, EC 1.1.1.49 (23); in vitro RNA synthesis (17);6PGDH, EC 1.1.1.44 (23); RNA (5).

RESULTS

Isolation of nuclei. Employing the experimentalprocedures described in Materials and Methods, nuclei

can be isolated from dry rice embryos in 30-50% yield.During centrifugation on a linear Percoll gradient (15-50%), more than 80% of the nuclei originally applied tothe centrifugation tube was recovered in the fractioncontaining up to 30% Percoll concentration (Fig. 1).Wehave found that discontinuous Percoll gradient cen-trifugation causes aggregation of nuclei accompanyingdrastic reduction of the yield. Consequently, a combina-tion of the step centrifugation on the 30%Percollcushion to remove starch granules and the differentialcentrifugation to removecytosolic components hasbeen used to achieve the satisfactory separation of purenuclear fractions in high yield.Microscopic examination of nuclei. Structural char-acterization of isolated nuclei was performed by exami-nation with both light and electron microscopes. As canbe seen from Fig. 2, the DAPI-stained fluorescent im-age (A) of the nuclei derived from the dry rice embryosis perfectly superimposable with their phase-contrast mi-croscopic image (C), indicating the absence of other con-taminating organelles. A magnified microphotograph(B) clearly shows the presence of dark regions, whichmaybe ascribed to the characteristic "dense chromatin"in the dry seed nucleus (3). Indeed, we have found thatthis image disappears during germination (not shown).The rice nucleus is 3-5 jum in diameter, relatively smal-ler than that of other plants, which is likely due to thesmaller genome size (6-9 x 108 bp) (24). It is also knownthat the rice nucleus cannot be stained with acetocar-mine solution, as in the case of Arabidopsis thaliana

50

- 4 0

1

- 3 0

1<｣

- 2 0

oo- 1 0 u .0 )Q _

10

Fraction numbeFig. 1. Distribution profile of nuclei from dry embryos on continu-ous Percoll gradient.Four ml of crude homogenates from dry rice embryos were layered on12ml of 15-50% continuous Percoll gradient and centrifuged at

6,800rpm for 30min in a Beckman SW-27.1 rotor. After fractio-nation of the centrifuged gradient (1 ml portion), the nuclei werecounted after DAPI staining (see Materials and Methods). Numbersof nuclei applied to the gradient were approximately 6.5 x 106 (100%).

88

Nuclei from Rice Embryos

50

Fig. 2. Light and electron microscopic image of isolated nuclei from dry rice embryos.A, B: DAPI stain, C: phase-contrast image, D: electron microscopic image. Size of bar; A and C: 50, B: 10, D: 2/um.

(28). Figure 2D shows the infrastructure of rice nucleiunder the electron microscope; the double envelopemembranestructure has been conserved. It will be alsonoted that the dense chromatin is clearly distinguisha-ble (cf. Fig. 2B).

Protein composition of nuclei derived from dry andgerminated rice embryos. The protein compositionwas compared between nuclei of dry and germinatedrice embryos (Fig. 3). The results of SDS-PAGEof thenuclear proteins showed that the electrophoretic mobili-ties of H3 and H4 were very similar to those of calf thy-mus histones, whereas HI, H2a and H2b appeared tobe different. It is well known that in contrast to the

highly conserved nature of H3 and H4, HI, H2a andH2b are variable amongvarious species (26). Further-more, in agreement with results of previous investiga-tions using wheat embryos (27), the histone content didnot differ much during the 2-d germination period ofrice embryos.

Solubilized nuclear protein components obtained bythe treatment with 0.5 M KC1 were subjected to SDS-PAGE analysis and results shown in Fig. 3B (Fl, 0-50% (NH4)2SO4 and F2, 50-100% (NH4)2SO4 saturatedfraction, respectively). It is evident that, under suchtreatment, histones are not extractable except HI pro-tein, and that the profiles of Fl and F2 are clearly distin-

89

J. Yamaguchi et al.

kDa

B) Nuclear ExtractskDa

dry 2-d

F1 F2 F1 F2

74.4"

49.6*

37.2.

24.8-IH1

74.4*

49.6-

37.2*

24.8*

1W

t «-ii£.a

12.4-

f

^H2bà"3fr «.-*- à"& Sk ~^ua-nt

12.4-

Fig. 3. SDS-PAGEof nuclei and nuclear extract prepared from dry and 2-d germinated rice embryos.A: Nuclei (20 jug protein) prepared from dry (lane dry) and germinated (lane 2-d). Rice embryos were subjected to SDS-PAGE. Lane H: histoneproteins from calf thymus. Symbols (*) indicate histone H3 and H4 from calf thymus, respectively. Arrow indicate rice histone H2a and H2b.B: Nuclear extract was prepared by a modification of the method described by Green et al. (9). Nuclei were sedimented at 3,000 x g for 5 min. Theresultant pellet was then gently resuspended on ice in 5.2 ml of nuclear lysis buffer (110 mMKC1, 15 mMHepes/KOH pH 7.6, 5 mMMgCl2, 1mMDTT, 5 //g/ml antipain, 5 jug/ml leupeptin) and transferred to a Beckman R-50 centrifuge tube, followed by the addition of 0.8 ml of 3 MKC1. The tube was gently rocked for 30 min at 4°C and then centrifuged at 45,000 rpm for 60 min. Proteins were precipitated by the addition of(NH4)2SO4 powder.

Lanes dry: nuclear extract prepared from dry embryos; lanes 2-d: from 2-d germinating embryos; lane Fl: proteins fractionated by 0-50%(NH4)2SO4 saturation; lane F2: by 50-100% (NH4)2SO4 saturation. Arrow indicate high mobility group (HMG)proteins of rice embyos.

guishable. It can be further shown that the low mol. wt.protein components increased during 2-d germinationof rice embryos. Although we have added 2-mercapto-ethanol, EDTA, leupeptin and antipain to the extrac-tion buffer, we cannot entirely excluded the possibilitythat the proteolytic digestion of nuclear proteins has oc-curred during the preparative step. It is most likely thatHMGproteins are present in the rice seed nuclei (see ar-row in Fig. 3B) (25).Purity of rice embryo nuclei. The purity of the iso-lated nuclei was examined by assaying several enzymeactivities of organelles that could possibly contaminatethe nuclear preparation. As summarized in Table la,the marker enzyme activities of cytosol, mitochondriaand plastids are barely detectable. However, high activ-ities of Cyt. c reductase, marker of ER, were found.Consistent with previous studies at both enzymic and ul-trastructural levels (6, 21), the results indicate thatsimilarities exist between the rER and nuclear enve-lopes. However, it was previously reported that the useof Triton X-100 to isolate nuclei from animal cells candiminish the contamination by ER membranes (21).

Table la. Enzyme activities in rice embryo nuclei.

specific activities(amole/min/mg protein)w h ole cells nu clei

A D H cyto sol 6. 6 2x 10 " n d

fum arase m itoch on drion 1 2 . 0 n d

G 6P D H plastid 3.17 x 10 -3 n d

6P G D H plastid 6.87 x lO "3 n d

catalase m icro bo dy 5 2 . 9 nd

cyt c reductase E R 2 34 1470

nd: not detectable

Table Ib. DNA, RNAand protein contentsOF NUCLEI FROM RICE EMBRYOS.

DNA(pg/nucleus)

RNADNA pro teinDNA

dry2-d

0.50

0.39

3.42

1.18

22.6

15.5

90

Nuclei from Rice Embryos

Table II. In vitro RNAsynthesis of rice embryo nucleiAND EFFECT OF INHIBITORS.

RNA synthesis C o ntrol

(cpm ) (% )

C on trol 3 ,25 1 100

+ a-Amanitin (4 ug/ml) 1,625 50

+Actinomycin D (4 ｫg/ml) 7 12 22

(40 ｣/g/ml) 689 2 1

- AT P , CT P , GT P 13 0.4

Nuclei were incubated with [3H]UTPin the reaction mixture accord-ing to the method described by Luthe and Quatrano (17). The reac-tion mixture contained 5.0 x 105 nuclei per assay.

Composition of isolated nuclei. The results ofDNA, RNAand protein analyses of isolated nuclei areshown in Table Ib. From the reported genome size ofrice (5.4x l08bp per diploid), we can estimate DNAcontent at 0.59 pg/nucleus. The DNAcontent obtainedfor the nucleus from the dry seed is close to this value,while the value is smaller at the 2-d germination stage.With respect to the ratios of RNA/DNAand protein/DNA, various data have been reported from differentlaboratories (ll, 21), but the values in the presentstudies are generally higher than the previously reportedones. The higher content of RNAand protein may con-ceivably reflect the highly purified state of the nuclearsamples used in the present investigation.Transcription in isolated rice nuclei. To determinethe transcriptional activity of nuclei isolated from riceembryos, assay of in vitro RNA synthesis was per-formed according to the procedure described by Lutheand Quatrano (17). The incorporation of [3H]UTP intotrichloroacetic acid-insoluble products was markedly in-hibited (80%) by actinomycin D and {50%) by a-amani-tin, respectively (Table II). The rate of transcription iscomparable with those reported using nuclei isolatedfrom some other plant (52% of that of wheat embryonuclei, 17), indicating that isolated nuclei from rice em-bryos are transcriptionally active.

DISCUSSION

Historically studies on the nucleus which plays a cen-tral role in the heredity of eukaryotic cells go back tothe 19th century. It will be remembered that the light mi-croscopic examination by Robert Brown of the plantmaterial Tradescantia first recognized the presence ofthe nucleus (Brownian motion). Since then numerouselaborate investigations have been carried out to exa-mine the complexultrastructural organization of the or-ganelle, and in parallel, vigorous efforts have been givento isolate nuclei from plant cells using various materialsas well as different experimental strategies (8, 10, 16, 17,29). With the recent advances in plant molecular biol-

ogy and molecular genetics, much attention has beenfocussed on the mutual interaction of multiple exact-ing regulatory sequences and trans-acting factors ofDNA-binding proteins to elucidate the mechanism oper-ating in gene expression. To this end, gel retardation as-says and DNaseI footprinting techniques are most fre-quently used. However, we must bear in mind that thesecommonrecombinant DNA techniques using the"naked" DNAdo not necessarily reflect the true pictureof gene regulation occurring in the chromatin in situ. In-deed manyrecent studies have disclosed the characteris-tic higher-order structural organization of the activechromatin, entailing the high transcriptional activities,and it is prerequisite to analyze the complex structure ofnuclei (chromatin) in eukaryotic cells.As can be readily surmised from the studies of steroidhormone (31), there is a good possibility that regulatoryproteins will be modified during the step of their entrythrough the nuclear envelopes. There exist multiple nu-clear matrix components such as protein kinase andphosphatase which can possibly activate elements in-volved in the transcriptional events. Therefore, it is ab-solutely necessary to characterize the structure and func-tion of nuclear components, e.g. nuclear envelope, nu-clear pore (pore complex), nuclear matrix, and chroma-tin as well as nuclear proteins such as histone, HMGprotein, and non-histone chromatin protein compo-nents. We believe that the isolation of intact nucleifrom rice seed embryos will provide us with a goodmeans of elucidating the mechanism(s) of gene expres-sion such as that typified by a-amylase gene which isknown to be activated during the step of germinationand also effected by the plant hormone, gibberellin (1,19). It is hoped that specific protein components presentin the nuclei and specifically involved in the expressionof a-amylase gene can nowbe examinedat the molecu-lar level.

Acknowledgement. The authors thank Dr. K. Kojima for his helpwith EM picture for nuclei and also Drs. L.M. Morgenthaler and M.Frehner for their critical reading of the manuscript and their valuablecomments. Wealso thank Dr. Imayasu at Gekkeikan Shuzo Co. Ltd.(Kyoto) for providing the rice embryos used in this study.

REFERENCES

1. Akazawa, T., Yamaguchi, J., and Hayashi, M. (1990). Ricea-amylase and gibberellin action-a personal view. In Gibberel-

lins (N. Takahashi, B.O. Phinney, and J. MacMillan, eds.).

Springer, New York, pp. 114-124.2. Comai, L. and Harada, J.J. (1990). Transcriptional activities

in dry seed nuclei indicate the timing of the transition from em-bryogeny to germination. Proc. Natl. Acad. Sci. USA, 87:

267 1-2674.

3. Deltour, R., Gautier, A., andFakan, J. (1979). Ultrastruc-tural cytochemistry of the nucleus in Zea mays embryos duringgermination. /. Cell Sci., 40: 43-62.

91

Dingwall, C. and Laskey, R.A. (1986). Protein import intothe cell nucleus. Ann. Rev. Cell Biol, 2: 367-390.

Fleck, A. and Munro, A.H.U. (1962). The precision of

ultraviolet absorption measurements in the Schmidt-Thannhau-ser procedure for nucleic acid estimation. Biochim. Biophys.

Ada, 55: 571-583.

Franke, W.W., Scheer, U., Krohne, G., and Jarasch, E.-D.(1981). The nuclear envelope and the architecture of the nu-

clear periphery. /. Cell Biol., 91: 39s-50s.Frehner, M., Pozueta-Romero, J., and Akazawa, T.

(1990). Enzyme sets of glycolysis, gluconeogenesis, and oxida-tive pentose phosphate pathway are not complete in nongreenhighly purified amyloplasts of sycamore {Acer pseudoplatanusL.) cell suspension cultures. Plant Physiol, 94: 538-544.Gallagher, T.F. and Ellis, R.J. (1982). Light-stimulated

transcription of genes for two chloroplast polypeptides in iso-lated pea leaf nuclei. EMBOJ., 1: 1493-1498.Green, P.J., Kay S.A., and Chua, N.-H. (1987). Sequence-

specific interactions of a pea nuclear factor with light-responsiveelements upstream of the rbcS-3A gene. EMBOJ., 6: 2543-

2549.

Guilfoyle, T.J., Suzich, J., and Lindberg, M. (1986). Syn-thesis of 5S rRNAand putative precursor tRNAs in nuclei iso-lated from wheat embryos. Plant Mol. Biol., 7: 95-104.Hamilton, R.H., Kunsch, U., and Temperli, A. (1972). Sim-ple rapid procedures for isolation of tobacco leaf nuclei. Anal.

Biochem., 49: 48-57.

Hatch, M.D. (1978). A simple spectrophotomeric assay forfumarate hydratase in crude tissue extracts. Anal. Biochem., 85:

271-275.

Hodges, T.K. and Leonard, R.T. (1974). Purification of aplasma membrane-bound ATPase from plant roots. Methods

EnzymoL, 32: 392-406.

Johnston, F.B. and Stern, H. (1957). Mass isolation of via-ble wheat embryos. Nature, 179: 160-161.Luck, H. (1965). Catalase. In Methods ofEnzymaticAnalysis(H. Bergmeyer, ed.). Academic Press, New York, pp. 885-894.

Luthe, D.S. and Quatrano, R.S. (1980). Transcription in iso-lated wheat nuclei. I. isolation of nuclei and elimination of endo-genous ribonuclease activity. Plant Physiol., 65: 305-308.

Luthe, D.S. and Quatrano, R.S. (1980). Transcription in iso-lated wheat nuclei. II. characterization of RNAsynthesized invitro. Plant Physiol., 65: 309-313.

Newmeyer, D.D. and Forbes, D.J. (1991). An N-ethylmale-imide-sensitive cytosolic factor necessary for nuclear protein im-

J. Yamaguchi et al.

port: requirement in signal-mediated binding to the nuclearpore. /. Cell BioL, 110: 547-557.

19. O'Neill, S.D., Kumagai, M.H., Majumdar, A., Huang, N.,Sutliff, T.D., and Rodriguez, R.L. (1990). The a-amylase

genes in Oryza sativa: characterization of CDNAclones andmRNAexpression during seed germination. MoI. Gen. Genet.,

221: 235-244.

20. Okamoto, K. and Akazawa, T. (1979). Enzymic mechanismof starch breakdown in germinatinng rice seeds. 7. Amylase for-mation in the epithelium. Plant Physiol., 63: 336-340.

21. Philipp, E.-L, Franke, W.W., Keenan, T.W., Stadler, J.and Jarasch, E.-D. (1976). Characterization on nuclear mem-branes and endoplasmic reticulum isolated from plant tissue. /.Cell BioL, 68: ll-29.

22. Reichelt, R., Holzenburg, A., Buhle, J., Jarnik, M.,

Engel, A., and Aebi, U. (1990). Correlation between struc-ture and mass distribution of the nuclear pore complex and ofdistinct pore complex components. /. Cell Biol., 110: 883-894.

23. Simcox, P.D., Reid, E.E., Canvin, D.T., and Dennis, D.T.(1977). Enzymes of the glycolytic and pentose phosphate path-ways in proplastids from the developing endosperm of Ricinuscommunis. Plant Physiol., 59: 1228-1232.

24. Sorbral, B.W.S., Honeycutt, R.J., Atherly, A.G., and

McClelland, M. (1990). Analysis of rice {Oryzasativa L.) gen-omeusing pulsed-field gel electrophoresis and rare-cutting re-

striction endonucleases. Plant Mol. Biol. Report., 8: 253-275.25. Spiker, S. (1984). High-mobility group chromosomal proteins

of wheat. /. Biol. Chem., 259: 12007-12013.26. Spiker, S. and Isenberg, I. (1977). Cross-complexing pattern

of plant histones. Biochemistry, 16: 1819-1826.27. Spiker, S. and Krishnaswamy, L. (1973). Constancy of

wheat histones during development. Planta (Berl.), 110: 71-76.28. Vlasak, J. (1981). Effect of different disintegration techniques

and media on yield and appearance of isolated nuclei. BiologiaPlantarum, 23: 406-413.

29. Watson, J.C. and Thompson, W.F. (1986). Purification andrestriction endonuclease analysis of plant nuclear DNA.Meth-ods EnzymoL, 118: 57-75.

30. Widholm, J.M. and Kishinami, I. (1988). Allyl alcohol selec-tion for lower alcohol dehydrogenase activity in Nicotiana

plumbaginifolia cultured cells. Plant Physiol., 86: 266-269.31. Yamamoto, K. (1985). Steroid receptor regulated transcrip-

tion of specific genes and gene networks. Annu. Rev. Genet.,19: 209-252.

{Receivedfor publication, October 1 7, 1991and in revised form, January 16, 1992)

92